Method for the selection of a long-term producing cell using histone acylation as markers

ABSTRACT

Herein is reported a method for determining methylation of a promoter nucleic acid operably linked to a nucleic acid encoding a polypeptide and thereby determining the long-term productivity of a cell. Also an aspect is a method for selecting a cell for producing a polypeptide by determining the methylation of the promoter nucleic acid operably linked to the structural gene encoding the polypeptide.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.15/311,460, filed Nov. 15, 2016, which is a 371 of InternationalApplication No. PCT/EP2015/060593 filed May 13, 2015 claiming priorityto European Application No. 14168896.0, filed May 19, 2014, which are/isincorporated herein by reference in its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Apr. 12, 2022, is namedSequence_Listing.txt and is 17,928 bytes in size.

The herein reported method is in the field of cell selection andpolypeptide expression/production. In more detail, herein is reported amethod for the selection of a long-term polypeptide expressing orsecreting cell based on the determination of histone acylation.

BACKGROUND OF THE INVENTION

DNA is a macromolecule that encodes the instructions of all known livingorganisms (Avery, O. T., et al., J. Exp. Med. 79 (1944) 137-158). Inhuman cells DNA has a length of approximately two meters and is mostlystored in the nucleus which has a diameter of 10 μm (Turner, B. M., Cell111 (2002) 285-291). To organize this amount of information the DNAneeds to be highly compacted. A group of conserved small basic proteinscalled histones form complexes with DNA to generate an ordered andcompact structure termed chromatin. Those positively charged proteinsinteract with the negatively charged phosphodiester backbone of the DNAdouble helix (Alberts, B. J. A., et al., Molecular Biology of the Cell;Meyers, R. A., Epigenetic Regulation and Epigenomics, WILEY-BLACKWELL,1; Olins, E. D., Nat. Rev. Mol. Cell Biol. (2003)). The four “core”histones H2A, H2B, H3 and H4 combine with DNA to form the basicrepeating unit of chromatin, called the nucleosome (Thomas, J. O. andKornberg, R. D., Proc. Natl. Acad. Sci. USA 72 (1975) 2626-2630). The225 kDa nucleosome core structure consists of approximately 147 bp ofDNA wrapped around a histone octamer, comprising two H2A/H2B dimers anda H3/H4 tetramer in 1.67 left-handed superhelical turns (Arents, G., etal., Proc. Natl. Acad. Sci. USA 88 (1991) 10148-10152; Arents, G. andMoudrianakis, E. N., Proc. Natl. Acad. Sci. USA 90 (1993), 10489-10493;Richmond, T. J., Scientist 13 (1999) 15-15). Depending on theaccessibility of DNA, chromatin is distinguished into two types. Highlycompacted heterochromatin, which is less accessible for transcription,and loosely-packed transcriptionally-active euchromatin. Facultativeheterochromatin can form anywhere in the nucleus, often localized topromoters, and is established either in a developmentally regulatedmanner or in response to environmental triggers (Chen, T. and Dent, S.Y., Nat. Rev. Genet. 15 (2014), 93-106).

The gradual loss of productivity in long-term culture is a common issuewith the development of manufacturing cell lines. The decrease ofrecombinant protein expression can be due to a loss of transgene copiesand/or silencing of the transgene promoter. Silencing is caused byepigenetic modifications of chromatin such as direct methylation ofpromoter DNA at CpG sites and posttranslational modifications ofhistones which are the major protein components of chromatin.Inactivating modifications of histones are counteracted by othermodifications that are activating.

Barnes, L. M., et al., report the molecular definition of predictiveindicators of stable protein expression in recombinant NSO myeloma cells(Biotechnol. Bioeng. 85 (2004) 115-121). The correlation reported byBarnes et al. is weak and not sufficient for a stability prediction.

In WO 2004/056986 means and methods for producing a protein throughchromatin openers that are capable of rendering chromatin moreaccessible to transcription factors are reported.

In WO 2011/128377 it has been reported that direct methylation of thehuman cytomegalovirus major-immediately-early promoter (hCMV MIE) can beused as early marker to predict production instability of recombinantCHO cell lines.

Osterlehner, A., et al., report promoter methylation and transgene copynumbers predict unstable protein production in recombinant Chinesehamster ovary cell lines (Biotechnol. Bioeng. 108 (2011) 2670-2681).

SUMMARY OF THE INVENTION

It has been found that the determination of the degree of methylation ofa specific CpG site in the promoter nucleic acid operably linked to astructural gene encoding a polypeptide in a cell or cell line used forthe production of the respective polypeptide in combination with thedetermination of histone acylation close to the promoter, i.e. in thepromoter chromatin, can be used to predict a decrease in productivityduring long-term cultivation. Additionally the copy number of the lightchain expression cassette integrated into the genome can be determined.

One aspect as reported herein is a method for selecting a cellclone/cell line, which comprises a nucleic acid comprising a structuralgene encoding a polypeptide operably linked to a promoter nucleic acid,comprising the following steps:

-   -   a) determining the (relative) level of histone 3 acetylation        relative to the level of histone 3 (H3ac/H3) close to the        promoter nucleic acid for a first multitude of cell clones/cell        lines, and    -   b) selecting a cell clone/cell line, which comprises a nucleic        acid comprising a structural gene encoding a polypeptide        operably linked to a promoter nucleic acid that has a (relative)        level of histone 3 acetylation relative to the level of histone        3 as determined in step a) of 0.1 or more.

One aspect as reported herein is a method for selecting a cellclone/cell line, which comprises a nucleic acid comprising a structuralgene encoding a polypeptide operably linked to a promoter nucleic acidcomprising the following steps:

-   -   a) determining for each cell clone/cell line of a first        multitude of cell clones/cell lines, whereby each clone/cell        line comprises a nucleic acid comprising a structural gene        encoding a polypeptide operably linked to a promoter nucleic        acid, the average (relative) level of histone 3 acetylation        relative to the level of histone 3 (H3ac/H3) close to the        promoter nucleic acid based on the (relative) level of histone 3        acetylation relative to the level of histone 3 (H3ac/H3)        determined in at least 10 cells obtained from a cultivation of        each cell clone/cell line, and    -   b) selecting a cell clone/cell line that has an average        (relative) level of histone 3 acetylation relative to the level        of histone 3 (H3ac/H3) of 0.1 or more.

One aspect as reported herein is a method for selecting a cellclone/cell line, which comprises a nucleic acid comprising a structuralgene encoding a polypeptide operably linked to a promoter nucleic acidthat has the nucleic acid sequence of SEQ ID NO: 01 comprising thefollowing steps:

-   -   a) determining the (relative) level of histone 3 acetylation        relative to the level of histone 3 (H3ac/H3) close to the        promoter nucleic acid that has the nucleotide sequence of SEQ ID        NO: 01 for each clone of a first multitude of cell clones/cell        lines, and    -   b) determining the methylation frequency of the CpG-site at        position 425 of SEQ ID NO: 01 for each clone of a second        multitude of cell clones/cell lines, and    -   c) selecting a cell clone/cell line, which comprises a nucleic        acid comprising a structural gene encoding a polypeptide        operably linked to a promoter nucleic acid that has the nucleic        acid sequence of SEQ ID NO: 01, that has a (relative) level of        histone 3 acetylation relative to the level of histone 3 as        determined in step a) of 0.1 or more, and that has a methylation        frequency of the CpG-site at position 425 as determined in        step b) of less than 5%.

One aspect as reported herein is a method for selecting a cellclone/cell line, which comprises a nucleic acid comprising a structuralgene encoding a polypeptide operably linked to a promoter nucleic acidthat has the nucleic acid sequence of SEQ ID NO: 01 comprising thefollowing steps:

-   -   a) determining for each cell clone/cell line of a first        multitude of cell clones/cell lines, whereby each clone/cell        line comprises a nucleic acid comprising a structural gene        encoding a polypeptide operably linked to a promoter nucleic        acid that has the nucleic acid sequence of SEQ ID NO: 01, the        average (relative) level of histone 3 acetylation relative to        the level of histone 3 (H3ac/H3) close to the promoter nucleic        acid that has the nucleotide sequence of SEQ ID NO: 01 based on        the (relative) level of histone 3 acetylation relative to the        level of histone 3 (H3ac/H3) determined in at least 10 cells        obtained from a cultivation of each cell clone/cell line,    -   b) determining for each cell clone/cell line of a second        multitude of cell clones/cell lines, whereby each clone/cell        line comprises a nucleic acid comprising a structural gene        encoding a polypeptide operably linked to a promoter nucleic        acid that has the nucleic acid sequence of SEQ ID NO: 01, the        average methylation frequency of the CpG-site at position 425 of        SEQ ID NO: 01 based on the methylation determined for at least        10 cells obtained from a cultivation of each cell clone/cell        line,    -   c) selecting a cell clone/cell line that has an average        (relative) level of histone 3 acetylation relative to the level        of histone 3 (H3ac/H3) of 0.1 or more, and that has a        methylation frequency at position 425 below 5%.

In one embodiment the (relative) level of histone 3 acetylation relativeto the level of histone 3 is 0.2 or more. In one embodiment the(relative) level of histone 3 acetylation relative to the level ofhistone 3 is 0.5 or more. In one embodiment the (relative) level ofhistone 3 acetylation relative to the level of histone 3 is 0.75 ormore. In one embodiment the (relative) level of histone 3 acetylationrelative to the level of histone 3 is 1.0 or more.

In one preferred embodiment the (relative) level of histone 3acetylation relative to the level of histone 3 is 0.5 or more.

In one embodiment the promoter nucleic acid has the sequence of SEQ IDNO: 01 or comprises a functional fragment thereof or a functionalvariant thereof. In one embodiment the CpG-site is at position 425 ofSEQ ID NO: 01 or a thereto corresponding position in the fragment orvariant thereof.

In one embodiment of all aspects the method further comprises thefollowing step:

ab) determining the (copy) number of stably integrated light chainexpression cassettes,

whereby step c) is:

selecting a cell clone/cell line that has a level of histone 3acetylation relative to the level of histone 3 as determined in step a)of more than 0.5, that has a methylation frequency of the CpG-site atposition 425 as determined in step b) of less than 5%, and that has a(copy) number of stably integrated light chain expression cassettes asdetermined in step ab) of 10 or less.

In one preferred embodiment the copy number of stably integrated lightchain expression cassettes as determined in step ab) is 6 or less.

In one embodiment the average (relative) level of histone 3 acetylationis the average (relative) level of histone 3 acetylation at the lysineresidues at position 4 and/or 9 and/or 14 and/or 18 and/or 27. In oneembodiment the lysine residues are at position 9 and/or 14 and/or 27.

In one embodiment of all aspects the method comprises as first step:

-   -   transfecting a population of cells with a nucleic acid        comprising a structural gene encoding a polypeptide operably        linked to a promoter nucleic acid that has the nucleic acid        sequence of SEQ ID NO: 01, and obtaining therefrom a first and a        second and optionally a third multitude of cell clones/cell        lines.

In one embodiment the (selected) cell line has a production rate after30-60 generations in cultivation of more than 60% of the production rateat the beginning of the cultivation.

One aspect as reported herein is a method for selecting a cellclone/cell line, which comprises a nucleic acid comprising a structuralgene encoding at least an antibody light chain operably linked to apromoter nucleic acid that has the nucleic acid sequence of SEQ ID NO:01 comprising the following steps:

-   -   a) determining the (relative) level of histone 3 acetylation        relative to the level of histone 3 (H3ac/H3) close to the        promoter nucleic acid that has the nucleotide sequence of SEQ ID        NO: 01 for each clone of a first multitude of cell clones/cell        lines, and    -   b) determining the (copy) number of stably integrated light        chain expression cassettes for each clone of a second multitude        of cell clones/cell lines, and    -   c) determining the methylation frequency of the CpG-site at        position 425 of SEQ ID NO: 01 for each clone of a third        multitude of cell clones/cell lines, and    -   d) selecting a cell clone/cell line, which comprises a nucleic        acid comprising a structural gene encoding a polypeptide        operably linked to a promoter nucleic acid that has the nucleic        acid sequence of SEQ ID NO: 01, i) that has a (relative) level        of histone 3 acetylation relative to the level of histone 3 as        determined in step a) of 0.1 or more, ii) that has a methylation        frequency of the CpG-site at position 425 as determined in        step b) of less than 5%, and iii) that has a (copy) number of        stably integrated light chain expression cassettes as determined        in step ab) of 10 or less.

One aspect as reported herein is a method for selecting a cellclone/cell line, which comprises a nucleic acid comprising a structuralgene encoding at least an antibody light chain operably linked to apromoter nucleic acid that has the nucleic acid sequence of SEQ ID NO:01 comprising the following steps:

-   -   a) determining for each cell clone/cell line of a multitude of        cell clones/cell lines, whereby each clone/cell line comprises a        nucleic acid comprising a structural gene encoding a polypeptide        operably linked to a promoter nucleic acid that has the nucleic        acid sequence of SEQ ID NO: 01, the average (relative) level of        histone 3 acetylation relative to the level of histone 3        (H3ac/H3) close to the promoter nucleic acid that has the        nucleotide sequence of SEQ ID NO: 01 based on the (relative)        level of histone 3 acetylation relative to the level of histone        3 (H3ac/H3) determined in at least 10 cells obtained from a        cultivation of each cell clone/cell line,    -   b) determining for each cell clone/cell line of the multitude of        cell clones/cell lines the (copy) number of stably integrated        light chain expression cassettes in at least 10 cells obtained        from a cultivation of each cell clone/cell line, and    -   c) determining for each cell clone/cell line of the multitude of        cell clones/cell lines, whereby each clone/cell line comprises a        nucleic acid comprising a structural gene encoding a polypeptide        operably linked to a promoter nucleic acid that has the nucleic        acid sequence of SEQ ID NO: 01, the average methylation        frequency of the CpG-site at position 425 of SEQ ID NO: 01 based        on the methylation determined for at least 10 cells obtained        from a cultivation of each cell clone/cell line,    -   d) selecting a cell clone/cell line i) that has an average        (relative) level of histone 3 acetylation relative to the level        of histone 3 (H3ac/H3) of 0.1 or more, ii) that has a        methylation frequency at position 425 below 5%, and iii) that        has a (copy) number of stably integrated light chain expression        cassettes as determined in step ab) of 10 or less.

In one embodiment the (relative) level of histone 3 acetylation relativeto the level of histone 3 is 0.2 or more. In one embodiment the(relative) level of histone 3 acetylation relative to the level ofhistone 3 is 0.5 or more. In one embodiment the (relative) level ofhistone 3 acetylation relative to the level of histone 3 is 0.75 ormore. In one embodiment the (relative) level of histone 3 acetylationrelative to the level of histone 3 is 1.0 or more.

In one preferred embodiment the (relative) level of histone 3acetylation relative to the level of histone 3 is 0.5 or more.

In one embodiment the average (relative) level of histone 3 acetylationis the average (relative) level of histone 3 acetylation at the lysineresidues at position 4 and/or 9 and/or 14 and/or 18 and/or 27. In oneembodiment the lysine residues are at position 9 and/or 14 and/or 27.

In one embodiment the promoter nucleic acid has the sequence of SEQ IDNO: 01 or comprises a functional fragment thereof or a functionalvariant thereof. In one embodiment the CpG-site is at position 425 ofSEQ ID NO: 01 or a thereto corresponding position in the fragment orvariant thereof.

In one preferred embodiment the (copy) number of stably integrated lightchain expression cassettes as determined in step b) is 6 or less.

In one embodiment the determining of the (relative) level of histone 3acetylation relative to the level of histone 3 (H3ac/H3) comprises thefollowing steps:

-   -   1) isolating chromatin from each of the cell clones/cell lines,    -   2) treating a first aliquot of the chromatin by an antibody that        is suitable to determine the (relative) level of histone 3        acetylation, such as e.g. a histone 3 acetylation specific        antibody, and forming an antibody-chromatin precipitate, and        treating a second aliquot of the chromatin by an antibody that        is suitable to determine the level of histone 3, such as e.g. a        histone 3 specific antibody, and forming and antibody-chromatin        precipitate,    -   3) amplifying genomic DNA from a third not-treated aliquot of        the chromatin and from the first and second treated aliquot with        real time quantitative PCR,    -   4) determining with the result obtained in step 3) the        (relative) level of histone 3 acetylation relative to the level        of histone 3.

In one embodiment the determining of the methylation frequency comprisesthe following steps:

-   -   1) isolating the DNA from each of the cell clones/cell lines,    -   2) performing for each isolated DNA individually a methylation        specific polymerase chain reaction,    -   3) determining with the results obtained in step 2) the        methylation frequency.

In one embodiment step 2) is

-   -   2) performing for each isolated DNA individually a polymerase        chain reaction with a methylation specific primer and a        universal primer.

In also an embodiment step 2) is

-   -   2) individually digesting the isolated DNA with a restriction        enzyme and performing a polymerase chain reaction for each of        the digested DNA with a methylation specific primer and a        universal primer.

In one embodiment the primers are independently of each other selectedfrom the group consisting of SEQ ID NO: 06 to 20.

In one embodiment a primers is selected from the group consisting of SEQID NO: 11, 14 and 15, the universal primer has the sequence of SEQ IDNO: 09, and a methylation specific primer is selected from the groupconsisting of SEQ ID NO: 17, 18 and 19.

In one embodiment the universal primers have the sequence of SEQ ID NO:09 and 11 and the methylation specific primers have the sequence of SEQID NO: 11 and 18.

In one embodiment the (relative) level of histone 3 acetylation relativeto the level of histone 3 (H3ac/H3) is normalized to a reference gene.In one embodiment the reference gene is Gusb.

In one embodiment the second multitude of cell clones/cell linescomprises at least one cell clone/cell line also comprised in the firstmultitude of cells clones/cell lines.

In one embodiment the second multitude of cell clones/cell lines isidentical to the first multitude of cells clones/cell lines.

In one embodiment the cell clone/cell line is a eukaryotic cellclone/cell line. In one embodiment the cell clone/cell line is amammalian cell clone/cell line. In one embodiment the cell clone/cellline is selected from CHO, BHK, HEK, and Sp2/0. In one embodiment thecell clone/cell line is a CHO cell clone/cell line. In one embodimentthe cell clone/cell line is a CHO K1 cell clone/cell line.

In one preferred embodiment the cell clone/cell line is a CHO cellclone/cell line.

In one embodiment the polypeptide is i) an antibody, or ii) an antibodyfragment, or iii) an antibody conjugate, or iv) an antibody light chainand an antibody heavy chain. In one embodiment the antibody is abispecific antibody.

One aspect as reported herein is a method for the production of apolypeptide comprising the following steps:

-   -   a) selecting a cell clone/cell line with a method as reported        herein,    -   b) cultivating the selected cell clone/cell line, and    -   c) recovering the polypeptide from the cultivation medium and/or        the cell clone/cell line and thereby producing the polypeptide.

In one embodiment the method comprises prior to step a) the followingsteps:

-   -   a-3) providing a mammalian, non-human cell,    -   a-2) transfecting the provided cell with a nucleic acid, which        comprises a nucleic acid comprising a structural gene encoding a        polypeptide operably linked to a promoter nucleic acid that has        the nucleic acid sequence of SEQ ID NO: 01,    -   a-1) i) optionally cultivating the transfected cell clone/cell        line in the presence of a selection agent, ii) single depositing        the transfected cells, and iii) cultivating the single deposited        transfected cells in the presence of a selection agent.

One aspect as reported herein is a method for the production of apolypeptide comprising the following steps:

-   -   a) selecting a cell clone/cell line with a method as reported        herein,    -   b) cultivating the selected cell/clone, and    -   c) recovering the polypeptide from the cultivation medium and/or        the cells and thereby producing a polypeptide.

In one embodiment the method comprises a further step

-   -   d) purifying the recovered polypeptide.

In one embodiment the method comprises prior to step a) the followingsteps:

-   -   a-3) providing a cell,    -   a-2) transfecting the provided cell with a nucleic acid        containing a structural gene encoding the polypeptide operably        linked to a promoter nucleic acid,    -   a-1) i) optionally cultivating and propagating the transfected        cell in the presence of a selection agent, ii) single depositing        the cells, and iii) cultivating the single deposited transfected        cells in the presence of a selection agent.

In one embodiment step a) comprises:

-   -   i) providing at least one cell comprising a nucleic acid        comprising a structural gene encoding the polypeptide operably        linked to a promoter nucleic acid,    -   ii) determining the methylation of the CpG-site at position 425        within the promoter nucleic acid of SEQ ID NO: 01, and    -   iii) selecting a cell producing a polypeptide wherein the        methylation determined in step b) is below a threshold value.

DETAILED DESCRIPTION OF THE INVENTION

Economic cell lines are required to provide high productivity and stableproduction levels during propagation from small to large scale. Decreaseof productivity during scale-up constitutes a serious risk during cellline development (Barnes, L. M., et al., Biotechnol. Bioeng. 81 (2003)631-639). One main reason for production instability is a reduction ofactive copy numbers over cell cycles, which might be attributed tochromosomal disruption/rearrangement as an inherent characteristic ofCHO cells (Kim, M., et al., Biotechnol. Bioeng. 108 (2011) 2434-2446.)and/or an induction by the gene amplification process (Kaufman, R. J.,et al., Mol. Cell. Biol. (1983) 699-711). The decrease of mRNA at aconstant number of copies of recombinant genes is another major cause ofproductivity drop (Barnes, L. M., et al., Biotechnol. Bioeng. 85 (2004)115-121; Chusainow, J., et al., Biotechnol. Bioeng. 102 (2009)1182-1196; Strutzenberger, K., et al., J. Biotechnol. 69 (1999)215-226). A reasonable explanation for this occurrence is epigeneticsilencing by promoter methylation (Osterlehner, A., et al., Biotechnol.Bioeng. 108 (2011) 2670-2681; Yang, Y., et al., J. Biotechnol. 147(2010) 180-185) and histone modifications as typified by deacetylationand specific methylation (Mutskov, V. and Felsenfeld, G., EMBO J. 23(2004) 138-149; Paredes, V., et al., Biotechnol. Lett. 35 (2013)987-993). Also, the recombinant sequence itself, the formation of tandemrepeats of sequence and the genomic location of transgene are proposedinitiators for gene silencing (Kaufman, W. L., et al., Nuc. Acids Res.36 (2008) e111). From this the influence of adjacent chromatin ontointegration sites is termed ‘position effect’ (Lattenmayer, C., et al.,Cytotechnol. 51 (2006) 171-182; Yin, Z., et al., Genet. Mol. Res. 11(2012) 355-369).

The promoter upstream of the recombinant gene initiates genetranscription and is able to affect gene expression level and stability(Kaufman, W. L., et al., Nuc. Acids Res. 36 (2008) eI11). The majorimmediate early gene promoter of the human cytomegalovirus (hCMV-MIE) iscommonly used to drive recombinant expression in mammalian cells forresearch and manufacturing to obtain high expression levels in transientand stable transfections (Boshart, M., et al., Cell 41 (1985) 521-530;Chapman, B. S., et al., Nuc. Acids Res. 19 (1991) 3979-3986; Foecking,M. K. and Hofstetter, H. Gene 45 (1986) 101-105; Wright, A., et al.,Hum. Gene Ther. 16 (2005) 881-892). Although the hCMV-MIE promoterprovides high gene expression levels, many studies have reported adecrease of productivity over long-term culture (Bailey, L. A., et al.,Biotechnol. Bioeng. 109 (2012) 2093-2103; Barnes, L. M., et al.,Biotechnol. Bioeng. 73 (2001) 261-270; He, L., et al., Biotechnol.Bioeng. 109 (2012) 1713-1722.). The silencing of the hCMV-MIE promoteris (in addition to the loss of copy numbers) largely attributed toepigenetic events of promoter DNA methylation and histone modification(Brooks, A. R., et al., J. Gene Med. 6 (2004) 395-404.; Kim, M., et al.,Biotechnol. Bioeng. 108 (2011) 2434-2446; Osterlehner, A., et al.,Biotechnol. Bioeng. 108 (2011) 2670-2681; Paredes, V., et al.,Biotechnol. Lett. 35 (2013) 987-993; Yang, Y., et al., J. Biotechnol.147 (2010) 180-185).

Chromatin is known to be involved in the regulation of gene expression.The modification of the DNA or of chromatin and/or chromatin-associatedproteins, such as histones, has an impact on chromatin structure andhence gene expression. DNA modification in mammalian cells can be bymethylation of cytosine residues in CpG dinucleotides. Histones,especially their N-terminal portion, can be modified by acetylation,methylation, phosphorylation or ubiquitinylation.

The nucleic acid content of a cell, i.e. its DNA, is present in the cellnucleus in compacted form together with histones. Histones pack andorder the DNA into nucleosomes. There are five main histone proteins inhumans. Histones are highly alkaline proteins. Histone H3 (histone 3)has a main globular domain and an N-terminal tail. It has a size of 137amino acid residues. Histones in general have the function of providinga core structure around which the DNA is located and regulate geneexpression.

Histones can be modified in many different ways, mainly occurring alongthe residues of the N-terminal amino acid sequence which ranges from 13to 40 amino acids in length depending on the specific histone. Thislarge number of modifications even increases due to the fact that somemodifications like lysine methylation comprise up to three differentstates (Kouzarides, T., Cell 128 (2007) 693-705). More than 100 histonemodifications have been discovered (Zentner, G. E. and Henikoff, S.,Nat. Struct. Mol. Biol. 20 (2013) 259-266). Acetylation and methylationof histone H3 and histone H4 tail residues are the best studiedmodifications. Overall, 14 distinctive types of modifications have beenfound (Dawson, M. A. and Kouzarides, T., Cell 150 (2012) 12-27) and arelisted in the following Table.

TABLE 1 Chromatin Modified Chromatin Modified Modifications ResiduesModifications Residues acetylation K-ac Proline P-cis > isomerizationP-trans methylation K-me1, K-me2, crotonylation K-cr (lysines) K-me3methylation R-me1, R-me2a, propionylation K-pr (arginines) R-me2sphosphorylation S-ph, T-ph butyrylation K-bu ubiquitinylation K-ubformylation K-fo sumoylation K-su hydroxylation Y-oh ADP ribosylationE-ar O-GlcNAcylation S-GlcNAc; (serine and T-GlcNAc threonine)deimination R > Cit K, lysine; R, arginine; S, serine; T, threonine; E,glutamic acid; P, proline; ac, acetylation; me, methylation; cit,citrulline; cr, crotonylation; pr, propionylation; bu, butyrylation; fo,formylation; oh, hydroxylation; a, asymmetric; s, symmetric; >,conversion. Adopted from (Dawson & Kouzarides, 2012).

The combination of these modifications, termed as PTM (posttranslational modification) motifs are rather associated with functionsthan the individual marks. Research into combination of modificationssuggests more than 200 different PTM motifs (Feller, C., et al., Mol.Cell 57 (2015) 559-571). Modifications are reversible.

It is known that methylation of histone H3 at lysine 9 (H3K9) and 27(H3K27) are modifications resulting in gene repression. Histonemodifications seem to provide labile transcriptional repression whereasDNA methylation being a highly stable silencing mark that is not easilyreversed (Cedar, H. and Bergman, Y., Nat. Rev. Gen. 10 (2009) 295-304).

Histone acetylation was discovered in 1961 as the first histonemodification (Phillips, D. M., Biochem. J. 87 (1963) 258-263). Earlystudies associate active transcribed genes with the hyperacetylation ofhistones, which indicates a function of acetylation in the transcriptionprocess (Allfrey, V. G., et al., Proc. Natl. Acad. Sci. USA 51 (1964)786-794). During S-phase a global increase of acetylation sites, such asH3K56ac, was observed followed by a decrease during G2-phase. This ledto the conclusion that histone acetylation might facilitate theincorporation of newly synthesized histones (Miller, K. M., et al., CellCycle 5 (2006) 2561-2565). In addition to their global role during DNAreplication, histone acetylation form specific genomic patternscorrelating with active transcription. Thereby heterochromatin regionsare hypoacetylated and euchromatin transcriptionally active genes arehighly acetylated (Kouzarides, T., Cell 128 (2007) 693-705). Acetylationpeaks are found at specific sites in the promoter close to transcriptionstart sites (TSS) (Wang, Z., et al., Nat. Genet. 40 (2008) 897-903). Thehistone lysine residues of the N-terminal tail of histone 3 at position4, 9, 14, 18 and of histone 4 at lysines 5, 8, and 12 are predominantlyprone to acetylation. Taken together, regulation of gene expression, DNAreplication, repair and recombination are influenced by differentacetylation states of histones (Dawson, M. A. and Kouzarides, T., Cell150 (2012) 12-27).

Histone acetyltransferases (HATS) generally mediate gene expression andtranscriptional activation (Cheung, P., et al., Cell 103 (2000)263-271). Using chromatin immunoprecipitation it has been found thatnon-methylated DNA is mostly assembled in nucleosomes that containacetylated histones, which are associated with open chromatin, whereasthe presence of methyl groups on identical DNA sequences correlates withassembly of nucleosomes containing non-acetylated histone H3 and H4leading to more compact chromatin (Cedar, H. and Bergman, Y., Nat. Rev.Gen. 10 (2009) 295-304; Eden, S., et al., Nature 394 (1998) 842-843;Hashimshony, T., et al., Nat. Gen. 34 (2003) 187-192). Histone H3 is themost excessively modified histone of the five naturally occurringhistones.

In regions of high transcriptional activity a high degree of histoneacetylation can be found. Histone acetylation is catalyzed by the enzymehistone-acetyl-transferase (HAT) which transfers the acetyl part ofacetyl-CoA to the F-amino group of specific histone lysine residues inthe N-terminal region. Histone acetylation takes exclusively place atlysine residues, such as e.g. H3K9 (lysine at position 9 of histone 3),H3K14, and H3K27 (Koch, C. M., et al., Gen. Res. 17 (2007) 691-707;Creyghton, M. P., et al., Proc. Natl. Acad. Sci. USA 107 (2010)21931-21936).

It is commonly observed during cell line development and in large scaleproduction that silencing of recombinant gene expression duringprolonged (large scale) host cell cultivation occurs. In mammalian cellsthis can be due e.g. to the formation of chromatin derivatives, whichprevents the transcription of the recombinant gene.

This results in a heterogeneous cell population after longer cultivationtimes, such as in large scale production (including seed trainfermentations and main fermentation). In this heterogeneous cellpopulation some cells continue to express high levels of the recombinantprotein of interest while other cells express low or even no protein ofinterest (see e.g. Martin, D. I. and Whitelaw, E., Bioessays 18, (1996)919-923; McBumey, M. W., et al., Exp. Cell. Res. 274 (2002) 1-8).

Production cell lines are in general descendants from a single parentcell. These cells are often scaled up during cultivation and cultivatedfor a long time in large scale fermentations (seed train and productionfermentations) resulting in cultivation volumes of up to 25,000 literand cell densities of often more than one million cells per milliliter.Such large scale fermentations can show dramatic reductions inproductivity (Migliaccio, A. R., et al., Gene 256 (2000) 197-214;Strutzenberger, K., et al., J. Biotechnol. 69 (1999) 215-226).

For the selection of production cell lines/cell clones, i.e. cellclone/cell lines that are intended to be used for the large scalerecombinant production of a polypeptide, such as e.g. an antibody, theproduction/expression stability, i.e. the loss of productivitygeneration by generation, of the cell clone/cell line is important.Thus, mammalian cell lines for recombinant protein production need tomaintain productivity over extended cultivation times. Generally theproduction stability of a cell clone/cell line is determined bycultivating the cell clone/cell line over a long period of time, i.e.multiple generations. At regular intervals the medium is diluted withfresh medium and the specific productivity per cell is determined basedon the product titer and the viable cell density. The change (normally areduction) of the specific productivity is indicative of long termproduction stability of the cell clone/cell line.

Long term stability studies are time and resource intensive, but arewidely performed to identify and eliminate unstable candidates duringcell line development. Depending on the target criteria the study covers30 to 60 cell divisions, which corresponds generally to 30 to 70 days.Thus, the required material and working time is pronounced. This can beseen even more by the fact that up to 75% of the tested cell lines/cellclones are not stable and, thus, a high number of cell lines/cell cloneshas to be assessed.

Beside the loss of the transgene production instability of manufacturingcell lines can be associated with methylation and silencing of theheterologous promoter used for the expression of the transgene (see e.g.Escher, G., et al. J. Lipid Res. 46 (2005) 356-365; Krishnan, M., etal., FASEB J. 20 (2006) 106-108; Yang, Y., et al., J. Biotechnol. 147(2010) 180-185). Promoter silencing can result from epigeneticmodification of the chromatin (combination or complex of DNA andproteins that make up the contents of the nucleus of a cell). This canbe the direct methylation of CpG dinucleotides within the promoter, suchas e.g. the human cytomegalovirus major-immediate-earlypromoter/enhancer (hCMV MIE), and/or the posttranslational modificationof histones. Besides inactivating histone modifications, such as themethylation of lysine 9 or 27 of histone 3, activating modifications,such as the methylation of lysine 4 of histone 3, or the globalacetylation of histone 3 and 4, are known (see e.g. Cedar, H. andBergman, Y., Nat. Rev. Genet. 10 (2009) 295-304).

The epigenetic modification of CpG methylation of the hCMV-MIE promoterwas used as an indicator for long-term production stability (seeOsterlehner et al.).

Herein local lysine acetylation (H3ac) as potential prediction markersof long-term transgene silencing was identified.

It has been found that the CpG dinucleotide at position 425 of the humanCMV major-immediate-early (hCMV) promoter/enhancer (SEQ ID NO: 01) isfrequently methylated in unstable antibody-producing Chinese hamsterovary (CHO) cell lines. A methylation-specific real-time qPCR has beenestablished to allow for the rapid and sensitive measurement of hCMV-MIEmethylation.

It has further been found that the posttranslational modification ofhistone adjacent to the promoter can be used as further marker toidentify stable expressing/producing cell lines. The presence of aninactivating modification is a marker that the respective cellclone/cell line is very instable and will show a decline in productivityduring the future course of the cultivation that is above average (longterm instable producing cell clone/cell line). On the other hand thepresence of an activating modification is a marker that the respectivecell clone/cell line is very stable and will show a decline inproductivity during the future course of the cultivation that is belowaverage (long term stable producing cell clone/cell line).

It has been found that by using a combination of both above markers,i.e. the methylation at position 425 of SEQ ID NO: 01 and the relativelevel of histone 3 acetylation relative to the level of histone 3(H3ac/H3) close to the promoter nucleic acid a further improvement inthe determination of long term stable producing recombinant celllines/cell clones can be achieved. A further improvement is possible ifalso the (copy) number of stably integrated light chain expressioncassettes is included in case of an antibody.

Thus, herein are reported methods for the selection of a cell clone/cellline as well as a method for the production of a polypeptide using sucha cell clone/cell line. The selected cell clone/cell line is a long-termproducing cell. Such a cell clone/cell line can be selected as reportedherein based i) on the relative level of histone 3 acetylation relativeto the level of histone 3 (H3ac/H3) close to the promoter nucleic acid,or ii) on the methylation of the human CMV major-immediate-early (hCMVMIE) promoter/enhancer at position 425 operably linked to the structuralgene encoding the polypeptide, or iii) on a combination of i) and ii).

A. Definitions

The term “almost” denotes that the value following this expression is acenter value with certain variability. In one embodiment the variabilityis of ±40% of the value, in another embodiment the variability is of±30%, and in a further embodiment the variability is of ±20%. Thus, theterm almost constant denotes that a value is in one embodiment in therange of from 60% to 140%, in another embodiment in the range of from70% to 130%, and in a further embodiment in the range of from 80% to120%.

The term “antibody” denotes a molecule comprising at least two so calledlight chain polypeptides (light chain) and two so called heavy chainpolypeptides (heavy chain). Each of the heavy and light chainpolypeptides comprises a variable domain (variable region) (generallythe amino terminal portion of the polypeptide chain) comprising bindingregions that are able to interact with an antigen. Each of the heavy andlight chain polypeptides also comprises a constant region (generally thecarboxy-terminal portion). The constant region of the heavy chainmediates the binding of the antibody i) to cells bearing a Fc gammareceptor (FcγR), such as phagocytic cells, or ii) to cells bearing theneonatal Fc receptor (FcRn) also known as Brambell receptor. It alsomediates the binding to some factors including factors of the classicalcomplement system such as component (C1q).

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

Depending on the amino acid sequence of the constant region of the heavychains, antibodies are divided in different classes: IgA class, IgDclass, IgE class, IgG class, and IgM class. Some of these classes arefurther divided into subclasses (isotypes), i.e. IgG in IgG1, IgG2,IgG3, and IgG4, or IgA in IgA1 and IgA2. According to the class to whichan antibody belongs the heavy chain constant regions are called α (IgA),δ (IgD), ε (IgE), γ (IgG), and (IgM), respectively. In one embodimentthe antibody is an antibody of the IgG class. In another embodiment theantibody has a human constant region or a constant region derived fromhuman origin. In a further embodiment the antibody is of the IgG4subclass or the IgG1, IgG2, or IgG3 subclass, which is modified in sucha way that no Fcγ receptor (e.g. FcγRIIIa) binding and/or no C1q bindingcan be detected. In one embodiment the antibody is of the human IgG4subclass or a mutated human IgG1 subclass. In one embodiment theantibody is of the human IgG1 subclass with mutations L234A and L235A.In another embodiment the antibody is in regard to Fcγ receptor bindingof IgG4 subclass or of IgG1 or IgG2 subclass, with a mutation in L234,L235, and/or D265, and/or contains the PVA236 mutation. In a furtherembodiment the antibody has a mutation selected from S228P, L234A,L235A, L235E, SPLE (S228P and L235E), and/or PVA236 (PVA236 means thatthe amino acid sequence ELLG (given in one letter amino acid code) fromamino acid position 233 to 236 of IgG1 or EFLG of IgG4 is replaced byPVA). In one embodiment the antibody is of the IgG4 subclass and has themutation S228P of IgG4, or the antibody is of the IgG1 subclass and hasthe mutations L234A and L235A.

The variable domain of an immunoglobulin's light or heavy chain in turncomprises different segments, i.e. four framework regions (FR) and threehypervariable regions (CDR).

The term “bisulfite treatment” denotes a reaction for the conversion ofcytosine bases in a nucleic acid to uracil bases in the presence ofbisulfite ions whereby 5-methyl-cytosine bases are not significantlyconverted. This reaction for the detection of methylated cytosine isdescribed in detail by Frommer et al. (Frommer, M., et al., Proc. Natl.Acad. Sci. USA 89 (1992) 1827-1831) and Grigg and Clark (Grigg, G. W.and Clark, S., Bioessays 16 (1994) 431-436; Grigg, G. W., DNA Seq. 6(1996) 189-198). The bisulfite reaction contains a deamination step anda desulfonation step which can be conducted separately orsimultaneously. The statement that 5-methyl-cytosine bases are notsignificantly converted shall only take the fact into account that itcannot be excluded that a small percentage of 5-methyl-cytosine bases isconverted to uracil although it is intended to convert only andexclusively the (non-methylated) cytosine bases.

The term “cell” denotes a cell into which a nucleic acid, e.g. encodinga, optionally heterologous, polypeptide, can be or isintroduced/transfected. The term “cell” includes both prokaryotic cells,which are used for propagation of plasmids, and eukaryotic cells, whichare used for the expression of a nucleic acid. In one embodiment thecell is a eukaryotic cell and in a further embodiment the eukaryoticcell is a mammalian cell. In another embodiment the mammalian cell isselected from the group of mammalian cells comprising CHO cells (e.g.CHO K1, CHO DG44), BHK cells, NSO cells, SP2/0 cells, HEK 293 cells, HEK293 EBNA cells, PER.C6® cells, and COS cells. As used herein, theexpression “cell” includes the subject cell and its progeny. Thus, theterm “cell” denotes the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understoodthat all progeny may not be precisely identical in DNA content, due todeliberate or inadvertent mutations. Variant progeny that have the samefunction or biological activity as screened for in the originallytransformed cell are included.

The term “CpG-site” denotes the dinucleotide CG within a nucleic acidthat can be recognized by the methylating enzymes of a cell and whereinthe cytosine can be converted to 5-methyl cytosine. In one embodimentthe CpG-site is within a promoter nucleic acid.

The term “expression cassette” denotes a construct that contains thenecessary regulatory elements, such as promoter and polyadenylationsite, for expression of at least the contained nucleic acid in a cell.

The term “expression plasmid” denotes a nucleic acid providing allrequired elements for the expression of the comprised structural gene(s)in a cell. Typically, an expression plasmid comprises a prokaryoticplasmid propagation unit, e.g. for E. coli, comprising an origin ofreplication, and a selection marker, an eukaryotic selection marker, andone or more expression cassettes for the expression of the structuralgene(s) of interest each comprising a promoter nucleic acid, astructural gene, and a transcription terminator including apolyadenylation signal. Gene expression is usually placed under thecontrol of a promoter nucleic acid, and such a structural gene is saidto be “operably linked to” the promoter nucleic acid. Similarly, aregulatory element and a core promoter nucleic acid are operably linkedif the regulatory element modulates the activity of the core promoternucleic acid.

The term “generation time” denotes the time required by a cell to divideand to produce a daughter cell. Thus, a cell that has divided once hasan age of one generation. The term “generation” denotes the number ofcell division of a cell.

The term “high frequency” denotes that at this methylation site thecytosine is methylated more often than at other methylation sites basedon the analysis of the methylation of a statistical significant numberof individual cells or DNA clones, respectively. This statisticalsignificant number is in one embodiment at least 10 individual cells orDNA clones, respectively, in a further embodiment at least 15 individualcells or DNA clones, respectively, and in another embodiment at least 20individual cells or DNA clones, respectively. In one embodiment atmaximum 400 cells or DNA clones, respectively, are analyzed.

The term “long-term producing cell” denotes a cell that produces apolypeptide, in one embodiment a heterologous polypeptide, whereby thespecific production rate of the cell is almost constant for at least 30generations. In one embodiment the long-term producing cell has aspecific production rate that is almost constant for at least 30generations, in another embodiment for at least 45 generations and in afurther embodiment for at least 60 generations. In one embodiment thelong-term producing cell has a specific production rate that is almostconstant for up to 60 generations, in a further embodiment for up to 75generations and in another embodiment for up to 90 generations.

The term “methylation” denotes a process within a cell that has beentransfected with a nucleic acid comprising a structural gene encoding apolypeptide operably linked to a promoter in which a cytosine of thepromoter nucleic acid is converted to 5-methyl cytosine. A promoternucleic acid in which at least one cytosine is converted to 5-methylcytosine is denoted as “methylated” nucleic acid.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein. In oneembodiment the antibody is a monoclonal antibody.

The term “operably linked” denotes a juxtaposition of two or morecomponents, wherein the components so described are in a relationshippermitting them to function in their intended manner. For example, apromoter and/or enhancer are operably linked to a coding sequence, if itacts in cis to control or modulate the transcription of the linkedsequence. Generally, but not necessarily, the DNA sequences that are“operably linked” are contiguous and, where necessary to join twoprotein encoding regions such as a secretory leader and a polypeptide,contiguous and in (reading) frame. However, although an operably linkedpromoter is generally located upstream of the coding sequence, it is notnecessarily contiguous with it. Enhancers do not have to be contiguous.An enhancer is operably linked to a coding sequence if the enhancerincreases transcription of the coding sequence. Operably linkedenhancers can be located upstream, within or downstream of codingsequences and at considerable distance from the promoter. Apolyadenylation site is operably linked to a coding sequence if it islocated at the downstream end of the coding sequence such thattranscription proceeds through the coding sequence into thepolyadenylation sequence. A translation stop codon is operably linked toan exonic nucleic acid sequence if it is located at the downstream end(3′ end) of the coding sequence such that translation proceeds throughthe coding sequence to the stop codon and is terminated there. Linkingis accomplished by recombinant methods known in the art, e.g., using PCRmethodology and/or by ligation at convenient restriction sites. Ifconvenient restriction sites do not exist, then syntheticoligonucleotide adaptors or linkers are used in accord with conventionalpractice.

The term “polypeptide” denotes a polymer consisting of amino acidsjoined by peptide bonds, whether produced naturally or synthetically.Polypeptides of less than about 20 amino acid residues may be referredto as “peptides”, whereas molecules consisting of two or morepolypeptides or comprising one polypeptide of more than 100 amino acidresidues may be referred to as “proteins”. A polypeptide may alsocomprise non-amino acid components, such as carbohydrate groups, metalions, or carboxylic acid esters. The non-amino acid components may beadded by the cell, in which the polypeptide is expressed, and may varywith the type of cell. Polypeptides are defined herein in terms of theiramino acid backbone structure or the nucleic acid encoding the same.Additions such as carbohydrate groups are generally not specified, butmay be present nonetheless.

The term “variant” of a promoter nucleic acid denotes that within thepromoter nucleic acid one or more nucleotides are changed withoutinterfering with the function of the promoter nucleic acid. Such achange may be for removing or introducing a restriction site.

The term “producing” denotes the expression of a structural geneinserted into an expression cassette in a cell. The term includes theprocesses of transcription and translation of nucleic acid. Producing isperformed in appropriate prokaryotic or eukaryotic cells and theexpressed, i.e. produced, polypeptide can be recovered from the cellsafter lysis or from the culture supernatant.

The term, promoter nucleic acid” denotes a polynucleotide sequence thatcontrols transcription of a gene/structural gene or nucleic acidsequence to which it is operably linked. A promoter nucleic acidincludes signals for RNA polymerase binding and transcriptioninitiation. The used promoter nucleic acid will be functional in thecell in which expression of the selected structural gene iscontemplated. A large number of promoter nucleic acids includingconstitutive, inducible and repressible promoters from a variety ofdifferent sources are well known in the art (and identified in databasessuch as GenBank) and are available as or within cloned polynucleotides(from, e.g., depositories such as ATCC as well as other commercial orindividual sources).

Typically, a promoter nucleic acid is located in the 5′ non-coding oruntranslated region of a gene, proximal to the transcriptional startsite of the structural gene. Sequence elements within promoter nucleicacids that function in the initiation of transcription are oftencharacterized by consensus nucleotide sequences. These elements includeRNA polymerase binding sites, TATA sequences, CAAT sequences,differentiation-specific elements (DSEs), cyclic AMP response elements(CREs), serum response elements (SREs), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF, AP2, SP1, cAMP response element binding protein (CREB) andoctamer factors. If a promoter nucleic acid is an inducible promoternucleic acid, then the rate of transcription increases in response to aninducing agent, such as a CMV promoter nucleic acid followed by twotet-operator site, the metallothionein and heat shock promoter nucleicacids. The rate of transcription is not regulated by an inducing agentif the promoter nucleic acid is a constitutively active promoter nucleicacid. Among the eukaryotic promoter nucleic acids that have beenidentified as strong promoter nucleic acids for expression are the SV40early promoter nucleic acid, the adenovirus major late promoter nucleicacid, the mouse metallothionein-I promoter nucleic acid, the Roussarcoma virus long terminal repeat, the Chinese hamster elongationfactor 1 alpha (CHEF-1), human EF-1 alpha, ubiquitin, and humancytomegalovirus major-immediate-early promoter nucleic acid (hCMV MIE).

The term “selection marker” denotes a nucleic acid that allows cellscarrying it to be specifically selected for or against, in the presenceof a corresponding selection agent. Typically, a selection marker willconfer resistance to a drug or compensate for a metabolic or catabolicdefect in the cell into which it is introduced. A selection marker canbe positive, negative, or bifunctional. A useful positive selectionmarker is an antibiotic resistance gene allowing for the selection ofcells transformed therewith in the presence of the correspondingselection agent, e.g. the antibiotic. A non-transformed cell is notcapable to grow or survive under the selective conditions, i.e. in thepresence of the selection agent. Negative selection markers allow cellscarrying the marker to be selectively eliminated. Selection markers usedwith eukaryotic cells include, e.g., the structural genes encodingaminoglycoside phosphotransferase (APH), such as e.g. the hygromycin(hyg), neomycin (neo), and G418 selection markers, dihydrofolatereductase (DHFR), thymidine kinase (tk), glutamine synthetase (GS),asparagine synthetase, tryptophan synthetase (selection agent indole),histidinol dehydrogenase (selection agent histidinol D), and nucleicacids conferring resistance to puromycin, bleomycin, phleomycin,chloramphenicol, Zeocin, and mycophenolic acid.

The term “short-term production rate” denotes the amount of polypeptideproduced by a single cell within one day as determined from the amountof polypeptide produced within a given time period and the viable celldensity, wherein the time period is short. In one embodiment theshort-term cultivation is for of from 2 to 20 days, in anotherembodiment for of from 4 to 15 days, and in still a further embodimentfor of from 10 to 14 days.

The term “specific production rate” or “production rate” denotes theamount of polypeptide produced by a single cell within one day asdetermined from the amount of polypeptide produced within a given timeperiod and the viable cell density. The specific production rate (SPR)can be calculated using the following formula:

SPR=P ₂ −P ₁/((D ₂ +D ₁)/2*Δt)  (Formula 2)

with

-   -   SPR [pg/cell/d]: specific production rate,    -   P₁ [μg/ml]: polypeptide concentration at the beginning of the        time period,    -   P₂ [μg/ml]: polypeptide concentration at the end of the time        period,    -   D₁ [cells/ml]: viable cell density at the beginning of the time        period,    -   D₂ [cells/ml]: viable cell density at the end of the time        period,    -   Δt [d]: duration of the time period.

The term “structural gene” denotes the region of a gene without a signalsequence, i.e. the coding region.

B. The Methods as Reported Herein

Cell lines/cell clones producing a polypeptide, i.e. cells transfectedwith a nucleic acid comprising an expression cassette containing astructural gene encoding a heterologous polypeptide, can be grouped indifferent classes: In a first class of cells the specific productionrate is almost constant over multiple generations. In contrast theretoin the second class of cells the specific production rate is decreasing,especially monotonously decreasing, with each generation over multiplegenerations. Without being bound by this theory the diminishingproductivity of polypeptide producing cells and cell lines,respectively, is caused at least in part by the steadily increasingmethylation and therewith inactivation/silencing of the promoter nucleicacid operably linked to the structural gene encoding the polypeptide ofinterest. Further the decrease can be by loss of copies of thestructural gene encoding the protein of interest.

The current invention is based, at least in part, on the finding thatthe CpG dinucleotide at position 425 of the human CMVmajor-immediate-early (hCMV MIE) promoter/enhancer (SEQ ID NO: 01) isfrequently methylated in unstable antibody-producing Chinese hamsterovary (CHO) cell lines. This can be used as marker for the prediction oflong-term recombinant production stability of a recombinant cellclone/cell line expressing a protein of interest encoded by a structuralgene operably linked to a hCMV-MIE promoter (e.g. of SEQ ID NO: 01).

The current invention is based, at least in part, on the finding thatthe posttranslational modification of histone adjacent to the promotercan be used as further marker to identify stable expressing/producingcell lines. The presence of an inactivating modification is a markerthat the respective cell clone/cell line is very instable and will showa decline in productivity during the future course of the cultivationthat is above average (long term instable producing cell clone/cellline). On the other hand the presence of an activating modification is amarker that the respective cell clone/cell line is very stable and willshow a decline in productivity during the future course of thecultivation that is below average (long term stable producing cellclone/cell line).

The current invention is based, at least in part, on the finding that byusing a combination of the methylation at position 425 of SEQ ID NO: 01and the relative level of histone 3 acetylation relative to the level ofhistone 3 (H3ac/H3) close to the promoter nucleic acid a furtherimprovement in the determination of long term stable producingrecombinant cell lines/cell clones can be achieved.

B.1. Promoter Methylation

It has been found that the presence of detectable methylation in thepromoter nucleic acid of the human CMV MIE promoter at position 425 thatis operably linked to the structural gene encoding a polypeptideprovides information regarding the long-term productivity of the cellline or cell clone, respectively.

Each promoter nucleic acid used for the expression of a structural genecomprises sites prone to methylation by the cell's enzymes into which ithas been introduced if the promoter nucleic acid is not shielded byprotective elements. A site amenable to methylation is termed CpG-siteand comprises/consists of the dinucleotide CG. But not all CpG-sites aremethylated with the same relative frequency—some of the CpG-sites aremethylated more often than others. It has been found that certain sitese.g. within the human CMV MIE promoter are methylated with differentfrequency and have a different impact on promoter silencing.

The following method can be used for identifying a CpG-site in a nucleicacid sequence:

-   -   1) providing a cell with a production rate of a polypeptide that        is after a cultivation time of 30 generations of the cell in the        absence of a selection agent less than 90% of the production        rate of the cell after the first generation of the cultivation,    -   2) separately isolating the DNA from at least 10 cells of a        cultivation of the cell of 1),    -   3) modifying the cytosine of the isolated DNA by bisulfite        treatment,    -   4) identifying a CpG site within the promoter nucleic acid        operably linked to the structural gene encoding the polypeptide        with a methylation frequency of at least 0.2 based on the DNA        obtained in step 3) and thereby identifying a CpG-site.

One criterion i.e. in one embodiment for an instable cell clone/cellline is a production rate of the cell clone/cell line after acultivation time of 30 to 60 generations of 60% or less than theproduction rate of the cell clone/cell line after the first generationof the cultivation.

In one embodiment the cell line has a production rate after 30-60generations in cultivation of more than 60% of the production rate atthe beginning of the cultivation.

The step of modifying the cytosine of the isolated DNA by bisulfitetreatment can comprise the following steps:

-   -   3-a) incubating the isolated DNA in the presence of sulfite ions        whereby the DNA is deaminated, and    -   3-b) incubating the deaminated DNA under alkaline conditions        whereby the deaminated DNA is desulfonated.

A method for obtaining a cell clone/cell line producing a polypeptide isa process comprising at least one transfecting step and at least oneselecting step including single cell depositing of successfullytransfected cells either directly after transfection or after growth inthe presence of a selection agent. In the selecting step cells areidentified based on their short-term specific production rate, i.e.based on the polypeptide concentration in the supernatant after ashort-term cultivation. Among the selected cells some have a specificproduction rate that is almost constant over multiple generations andothers have a specific production rate that is monotonously decreasingover multiple generations. Thus, with generally applied (short term)selection criteria no specific selection of a cell or cells with astable long-term productivity can be made.

In the methods as reported herein any cell clone/cell line obtained bytransfection with an expression plasmid comprising an expressioncassette comprising a promoter nucleic acid operably linked to astructural gene encoding a polypeptide of interest to be produced by thetransfected cell can be analyzed. The expression plasmid generallycomprises also a selection marker. Thus, the cells are cultivated in thepresence of a selection agent after the transfecting step and prior tothe selecting step. Alternatively the method can comprise cultivatingthe cells without prior single cell deposition or limited dilution, i.e.as pool, in the presence of a selection agent. Further alternatively themethod can comprise cultivating the cells after single cell depositionor limiting dilution.

After the pool cultivation/selection a single cell deposition has to beperformed. If the single cell deposition is performed after a poolcultivation step the cells are also further cultivated after the singlecell deposition.

In order to identify cells lines or cell clones with a specificproduction rate that is almost constant over multiple generations thepolypeptide concentration in the supernatant and the viable cell densityhave to be determined at defined cultivation times in a long-termcultivation over multiple generations. Methods suitable therefore areknown to a person skilled in the art, such as ELISA and FACS,respectively.

A CpG-site with a high methylation frequency can be identified bybisulfite treatment of single stranded DNA, e.g. at pH 5, withsucceeding alkaline desulfurization. Herein methylated andnon-methylated CpG-sites can be discriminated. Under the specifictreatment conditions cytosine but not 5-methyl-cytosine is deaminated atposition 4 of the N-heterocycle and converted to uracil. ComplementaryDNA strands are converted into two strands, strand A and strand B, whichare no longer complementary. The determination can be based on any ofthese strands.

This long-term cultivation has to be performed only once for thepromoter nucleic acid or combination of promoter nucleic acid and cellline. If the same promoter nucleic acid or combination of cell andpromoter is used a second time the selection can be based on the alreadycollected data

A number of techniques can be applied to reveal sequence differencesbetween methylated and non-methylated alleles after bisulfite treatment.For example the sequence of interest (either strand A or strand B) canbe amplified by PCR under non-methylation specific conditions, i.e. withprimer not sensitive to methylated sites, and subsequently analyzed bymethods such as DNA sequencing (with or without cloning), highresolution melting point analysis, or microarray analysis. In anotherembodiment a quantitative PCR (qPCR) with methylation sensitive ormethylation specific primer or probes can be performed. In analternative approach methylated DNA is precipitated with 5-methylcytosine specific antibodies followed by a quantitative polymerase chainreaction.

Methylation specific PCR (MSP) can also be used to address the sequenceof bisulfite treated DNA directly without previous PCR amplification ofthe region of interest. Primers used in MSP shall comprise one or moreCpG-sites. They are either complementary to unconverted5-methyl-cytosine for the detection of methylated DNA or complementaryto uracil converted from cytosine for the detection of non-methylatedDNA.

The methylation of the CpG-site is determined for a number of cells. Inone embodiment the number of cells is at least 10, in another embodimentat least 15, and in a further embodiment at least 20. Afterwards themethylation frequency for the CpG-site is calculated, i.e. the number ofcells methylated at that CpG-site divided by the total number of cellsanalyzed is calculated. A CpG-site with a high methylation frequency isa CpG-site that has a methylation frequency of at least 0.2, in oneembodiment of at least 0.25, in one embodiment of at least 0.4, and inone preferred embodiment of at least 0.5.

It has been found that even low levels of methylated promoter nucleicacid, i.e. above a predetermined threshold value, in a cell or cell lineused for the production of a polypeptide can be used to predict adecrease in productivity during long-term cultivation.

In FIG. 1 the number of methylated CpG site of the same promoter nucleicacid obtained from different cells is shown.

The human CMV MIE promoter as a nucleic acid sequence as depicted in thefollowing (CpG sites are underlined):

(SEQ ID NO: 01) ATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATAT ATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCAT TGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGA GTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGAC GTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGC AGTACATCTACGTATTAGTCATCGCTATTAGCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGG ATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCAC CAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTG TACGGTGGGAGGTCTATATAAGCAGAGCTCCGTTTAGTGAACG.

In SEQ ID NO: 01 thirty-three CpG sites are present, which are potentialsites for nucleic acid methylation. With the method as outlined abovecytosine residues within the human CMV MIE promoter that arepredominantly methylated can be identified.

The strand A with all CpG-sites preserved has the nucleotide sequence

ATGTTGATATTGATTATTGATTAGTTATTAATAGT AATTAATTACGGGGTTATTAGTTTATAGTTTATATATGGAGTTTCGCGTTATATAATTTACGGTAAATGG TTCGTTTGGTTGATCGTTTAACGATTTTCGTTTATTGACGTTAATAATGACGTATGTTTTTATAGTAACG TTAATAGGGATTTTTTATTGACGTTAATGGGTGGAGTATTTACGGTAAATTGTTTATTTGGTAGTATATT AAGTGTATTATATGTTAAGTACGTTTTTTATTGACGTTAATGACGGTAAATGGTTCGTTTGGTATTATGT TTAGTATATGATTTTATGGGATTTTTTTATTTGGTAGTATATTTACGTATTAGTTATCGTTATTAGTATG GTGATGCGGTTTTGGTAGTATATTAATGGGCGTGGATAGCGGTTTGATTTACGGGGATTTTTAAGTTTTT ATTTTATTGACGTTAATGGGAGTTTGTTTTGGTATTAAAATTAACGGGATTTTTTAAAATGTCGTAATAA TTTCGTTTTATTGACGTAAATGGGCGGTAGGCGTGTACGGTGGGAGGTTTATATAAGTAGAGTTTCGTTT AGTGAACG

(SEQ ID NO: 02) and the completely deaminated strand A has thenucleotide sequence

ATGTTGATATTGATTATTGATTAGTTATTAATAGT AATTAATTATGGGGTTATTAGTTTATAGTTTATATATGGAGTTTTGTGTTATATAATTTATGGTAAATGG TTTGTTTGGTTGATTGTTTAATGATTTTTGTTTATTGATGTTAATAATGATGTATGTTTTTATAGTAATG TTAATAGGGATTTTTTATTGATGTTAATGGGTGGAGTATTTATGGTAAATTGTTTATTTGGTAGTATATT AAGTGTATTATATGTTAAGTATGTTTTTTATTGATGTTAATGATGGTAAATGGTTTGTTTGGTATTATGT TTAGTATATGATTTTATGGGATTTTTTTATTTGGTAGTATATTTATGTATTAGTTATTGTTATTAGTATG GTGATGTGGTTTTGGTAGTATATTAATGGGTGTGGATAGTGGTTTGATTTATGGGGATTTTTAAGTTTTT ATTTTATTGATGTTAATGGGAGTTTGTTTTGGTATTAAAATTAATGGGATTTTTTAAAATGTTGTAATAA TTTTGTTTTATTGATGTAAATGGGTGGTAGGTGTGTATGGTGGGAGGTTTATATAAGTAGAGTTTTGTTT AGTGAATG

(SEQ ID NO: 03). The strand B with all CpG-sites preserved has thenucleotide sequence

CGTTTATTAAACGGAGTTTTGTTTATATAGATTTT TTATCGTATACGTTTATCGTTTATTTGCGTTAATGGGGCGGAGTTGTTACGATATTTTGGAAAGTTTCGT TGATTTTGGTGTTAAAATAAATTTTTATTGACGTTAATGGGGTGGAGATTTGGAAATTTTCGTGAGTTAA ATCGTTATTTACGTTTATTGATGTATTGTTAAAATCGTATTATTATGTTAATAGCGATGATTAATACGTA GATGTATTGTTAAGTAGGAAAGTTTTATAAGGTTATGTATTGGGTATAATGTTAGGCGGGTTATTTATCG TTATTGACGTTAATAGGGGGCGTATTTGGTATATGATATATTTGATGTATTGTTAAGTGGGTAGTTTATC GTAAATATTTTATTTATTGACGTTAATGGAAAGTTTTTATTGGCGTTATTATGGGAATATACGTTATTAT TGACGTTAATGGGCGGGGGTCGTTGGGCGGTTAGTTAGGCGGGTTATTTATCGTAAGTTATGTAACGCGG AATTTTATATATGGGTTATGAATTAATGATTTCGTAATTGATTATTATTAATAATTAGTTAATAATTAAT GTTAATAT

(SEQ ID NO: 04) and strand B in completely deaminated form has thenucleotide sequence

(SEQ ID NO: 05) TGTTTATTAAATGGAGTTTTGTTTATATAGATTTTTTATTGTATATGTTTATTGTTTATTTGTGTTAATG GGGTGGAGTTGTTATGATATTTTGGAAAGTTTTGTTGATTTTGGTGTTAAAATAAATTTTTATTGATGTT AATGGGGTGGAGATTTGGAAATTTTTGTGAGTTAAATTGTTATTTATGTTTATTGATGTATTGTTAAAAT TGTATTATTATGTTAATAGTGATGATTAATATGTAGATGTATTGTTAAGTAGGAAAGTTTTATAAGGTTA TGTATTGGGTATAATGTTAGGTGGGTTATTTATTGTTATTGATGTTAATAGGGGGTGTATTTGGTATATG ATATATTTGATGTATTGTTAAGTGGGTAGTTTATTGTAAATATTTTATTTATTGATGTTAATGGAAAGTT TTTATTGGTGTTATTATGGGAATATATGTTATTATTGATGTTAATGGGTGGGGGTTGTTGGGTGGTTAGT TAGGTGGGTTATTTATTGTAAGTTATGTAATGTGGAATTTTATATATGGGTTATGAATTAATGATTTTGT AATTGATTATTATTAATAATTAGTTAATAATTAATGTTAATAT.

In FIG. 4 the frequency of methylation at individual CpG-sites indifferent cell lines is shown. The numbers have been determined byanalyzing 19 to 22 different clones obtained from different CHO parentcell lines after transfection with a plasmid comprising an expressioncassette for expressing a polypeptide of interest. Shown is themethylation pattern of single DNAs (bottom) and the frequency ofmethylation at single CpG sites (top) for each cell line. Cell lineK18.1 for example is highly methylated (FIG. 4A). The frequency ofmethylation is not equal at the different CpG sites but seems to havecenters in 3 clusters, i.e. at the 5′-end, the 3′-end, and aroundposition (or nucleotide, respectively) 400. Fourteen out of 22 sequencedinserts had a cytosine at position 425. The methylation of the promoternucleic acid in cell line 43-16 A10 is shown in FIG. 4E. Thedistribution of methylation is similar to the distribution observed withcell line K18.1. As with K18.1 the position 425 was methylated mostoften: five of 20 inserts sequenced contained a cytosine in thisposition.

In three other analyzed cell lines cytosine is detected sporadically,i.e. as single events, at different CpG sites (FIGS. 4B, 4C and 4D). Toobtain statistically significance for cell lines with a low overallmethylation frequency sequencing of hundreds of inserts would berequired. Additionally these single events may also represent falsepositive events due to incomplete cytosine deamination rather thanactual promoter methylation.

For reliable determination of CpG position specific methylation amethylation specific PCR method has been developed. For the methylationspecific PCR primers as shown in the following Table 2 can be used.These primers either alone or in combination are also aspects asreported herein.

TABLE 2 Primers that can be used in methylation specific PCR. primermethy- no. lation SEQ (direc- spe- ID tion) cific nucleotide sequenceNO: 227 no ATGTTGATATTGATTATTGATT 06 (forward) AG 228 noTATGGGATTTTTTTATTTGGTA 07 (forward) GT 229 no ACTCCTCTCCCAAAACTAAAT 08(reverse) CTA 237 no CCAAAACAAACTCCCATTAAC 09 (reverse) 238 noGGGGTTATTAGTTTATAGTTTA 10 (forward) TA 239 no TGGTATTATGTTTAGTATATGA 11(forward) TTTTAT 240 no GGATTTTTTTATTTGGTAGTAT 12 (forward) ATT 254 yesAAATCCCCGTAAATCAAACCG 13 (reverse) 263 no GGGATTTTTTTATTTGGTAGTA 14(forward) TATT 264 no TATGGGATTTTTTTATTTGGTA 15 (forward) TGA 265 yesATCCCCGTAAATCAAACCG 16 (reverse) 266 yes TCCCCGTAAATCAAACCG 17 (reverse)267 yes CCCCGTAAATCAAACCG 18 (reverse) 268 yes CCCGTAAATCAAACCGC 19(reverse) 262 yes AAATCCCCRTAAATCAAACCG 20 (reverse)

In the course of primer evaluation it has been found that methylationspecific primer pairs, that are highly selective for deaminated CMWpromoter DNA with a cytosine at position 425, differ in their properties(see FIG. 6 ). In one embodiment the primers for the methylationspecific PCR have the nucleotide sequence of SEQ ID NO: 14 and SEQ IDNO: 18.

Thus, in one embodiment of the methods as reported herein is thepromoter nucleic acid the human CMV promoter nucleic acid of SEQ ID NO:01. In one embodiment the CpG-site with high methylation frequency isthe CpG-sites at position (bp) 425 of SEQ ID NO: 01.

TABLE 3 Expected results for methylation specific (MSP) primer pairs anduniversal primer pair in qPCR. Template #11 #62 #01 #04 position 425 T CC T position 437 T C T C Amplification methylation specific primer pair− + + − universal primer pair + + + +

The universal primer pair should amplify all four templates whereas theMSP primer pair should selectively amplify template #62 (SEQ ID NO: 22)and template #01 (SEQ ID NO: 23). The ΔCp value should be as small aspossible between the MSP primer pair and the universal primer pair ontemplate #62 and template #01. By contrast, Cp values obtained with theMSP primer pair on templates #11 (SEQ ID NO: 21) and #04 (SEQ ID NO: 24)should be as high as possible, i.e. ΔCp compared to amplification withthe universal primer pair should be maximal.

The methylation specific primer should be able to detect 5-methylcytosine at position 425 selectively albeit two further methylationpositions are present at position 416 and 437.

The determination of the methylation is possible with a frequency ofmethylation of from 1% to 100%.

Comparable results with respect to the methylation extent were observedwith a methylation specific PCR and by cloning and sequencing, wherebythe methylation specific PCR is much more sensitive. Cell lines with adecreasing productivity in long-term production have a methylation atposition 425 with a methylation frequency above a threshold value. Thethreshold value is in one embodiment twice the background noise of thedetermination method. A cell line with long-term stable productivity hasa frequency of methylation at the CpG site that is below the thresholdvalue.

For high-resolution melting point analysis the promoter nucleic acid isamplified starting from position 334 up to position 487, i.e. 154 bps.An exemplary melting point analysis is shown in FIG. 10A and its firstderivative in FIG. 10B. It can be seen that with a high-resolutionmelting point analysis a methylated promoter nucleic acid (template #16,SEQ ID NO: 25) can be distinguished from a non-methylated promoternucleic acid (template #11). The methylated promoter nucleic acidfragment can be detected at a relative frequency of 50% or more. Thenon-methylated promoter fragment can be detected at a relative frequencyof 10% or more.

This data shows that the production stability of recombinant CHO celllines that contain recombinantly introduced genes whose expression isdriven by the human CMV major-immediate-early promoter/enhancer can bepredicted by measuring the methylation status of the cytosine atposition 425.

Thus, it has been found that the determination of C425 methylation canbe used as a predictive marker to determine the stability of polypeptideexpression in generated recombinant cell clones. This allows theselection of stable clones with stable productivity during cell linedevelopment. It has further been found that C425 methylation of 5% orless is a suitable criterion for the selection of stable cell clones(this takes into account the threshold of the used detection method). Ithas also been found that the fraction of cell clones that are falselypredicted as stable (false negative cell clones) can be reduced bycultivating them for some time in the absence of MTX before testing.

Thus, herein is reported a method for the enrichment of long term(stable) recombinant polypeptide expressing cells lines from apopulation of transfected cells by selecting cells having a relativepromoter methylation at position 425 of SEQ ID NO: 01 of 5% or less.This relative methylation frequency can be determined using amethylation-specific qPCR method as reported herein with the primers asreported herein.

Having established a sensitive and accurate PCR method to quantifymethylation of hCMV-MIE nucleotide at CpG site 425, the methylation inrecombinant CHO cell lines K18.1, 43 16 A10 and G45-2 has been assessed.The bisulfite-treated DNA that had been analyzed by sequencing was usedas template in methylation-specific real time qPCR, either directly orafter PCR amplification of the complete hCMV-MIE region (FIGS. 8A and8B). The two assay set-ups provided comparable results.

More importantly, the results correlated well with the results ofbisulfite sequencing. This demonstrates that the CpG site 425methylation-specific qPCR assay can be used to measure hCMV-MIEmethylation at CpG site 425 in recombinant CHO cell lines and can beused without previous PCR amplification of the target DNA.

In principal, other CpG sites within the CMV major-immediate-earlypromoter/enhancer DNA could be explored to predict productioninstability. However, methylation at C425 was found to be approximately5-fold higher than the average methylation at all CpG sites. Moreoversome sites were not methylated at all even in highly methylated cellclones, e.g. C280 and C289 (FIGS. 4F, 4J and 13 ). By chosen the righti.e. frequently modified CpG site for promoter methylation analysis theassay becomes more sensitive. Significant methylation of clones G25-17,G25-10 and 43-16 A10 which is about 10% would likely be missed, if a CpGsite was randomly chosen for analysis.

The stability prediction by addressing the methylation status of arelevant CpG sites within a promoter nucleic acid can be used also withother heterogenic promoter nucleic acids.

To evaluate a potential correlation of early promoter methylation andproduction instability, CpG site 425 methylation at the start of astability study was plotted against the relative alteration of qP in thepresence or in the absence of a selection agent (MTX). The correlationplots are shown in FIGS. 11A (with MTX) and 11B (without MTX). For theevaluation, the plot areas were divided into four compartments withlimits at 5% methylation—equaling the two to three-fold background ofthe methylation measurement, i.e. the limit of detection—and 40%decrease in qP—representing the acceptance limit of production stability(this is also an embodiment of the method as reported herein). Thenumber of clones in the different compartments was determined.Contingency analysis of stability status by methylation status using aPearson chi square test demonstrated a significant association with ap-value of 0.05 for cultivation with MTX and a trend with a p-value of0.13 for cultivation without MTX. It turned out that the majority ofclones with less than 5% methylation at CpG site 425 were found in thefraction of stable clones (less than 40% decrease in qP with or withoutMTX, shown in upper left compartments).

Thus, summarizing the above, loss of productivity during long termcultivation and scale-up is a major risk in the development ofmanufacturing cell lines. Therefore, there exists a need for molecularmarkers of production instability that can be rapidly and easilydetermined and examined. It has been found that promoter methylation canbe employed to predict a loss of productivity in recombinant CHO celllines. To assess this, DNA methylation of 33 CpG sites within a 603 bpregion of the widely used hCMV-MIE promoter/enhancer was analyzed. Theoverall methylation level of the region investigated varied betweenapproximately 1% and 18% of all CpG sites. One percent apparentmethylation represents the technically achievable background resultingfrom incomplete deamination of non-methylated cytosines. Moreover,within methylated promoters, the level of methylation greatly variesbetween individual CpG sites and accumulates in three clusters with amaximum at CpG site 425. Methylation at site CpG 425 is approximately5-fold higher than the average degree of methylation of all other CpGsites. On the other hand, some CpG sites appear to be completelynon-methylated, even in highly methylated cell lines. The overallmethylation of hCMV-MIE, as well as the distribution of methylationbetween individual CpG sites, can vary considerably between cell typesand tissues (see e.g. Kong, Q., et al., PLoS One 4 (2009) e6679;Krishnan, M., et al., FASEB J. 20 (2006) 106-108; Mehta, A. K., et al.,Gene 428 (2009) 20-24).

It has been found that the dominant methylation of CpG site 425 issuited as marker for methylation of hCMV-MIE. It has been established aCpG site 425 methylation-specific qPCR as a fast and sensitive methodwith medium throughput. When analyzing a large number of cell lines byCpG site 425 methylation-specific qPCR, it has been found that themajority of unstable producers displayed more than 5% methylation at CpGsite 425, even before long-term cultivation, whereas the majority of thestable producers showed less than 5% methylation at this site.

Early methylation of CpG site 425 was exclusively found with clonescarrying more than ten copies of the heterologous plasmid. Previousreports have provided some evidence that tandem repeats of multipletransgene copies are more susceptible to methylation and silencing inmammalian cells (Garrick, D., et al., Nat. Genet. 18 (1998) 56-59,McBurney, M. W., et al., Exp. Cell Res. 274 (2002) 1-8).

B.2. Histone Acylation

The relative amounts of histone modifications close to the hCMV-MIEpromoter were determined. The H3ac and H3K4me3 marks (activating marks)and H3K9me3 as well as H3K27me3 marks (repression marks) were examined.Those histone modifications were examined at positions 404 to 507 of SEQID NO: 01 (human CMV-MIE promoter sequence; tacatca atgggcgtggatagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagtttgttttggcac caaaatcaac gggactt) which indicates a sequence from −97 to−200 bp upstream of transcription start site and contains the CpG siteat position C-425 (i.e. −179 bp upstream of transcription start site)and which can be amplified with primer pair 396/397.

Levels of relative histone 3 acetylation relative to the level ofhistone 3 close to human CMV major-immediate-early promoter/enhancerfragment was investigated as prediction markers for production stabilitywith and without selection agent.

Two exemplary frozen cell lines from the start point (Primary Seed Bank)of cultivation were thawed and the relative amounts of specified histonemodifications close to the hCMV-MIE promoter were determined. Project Tand project H were independently executed and each cell line wascultivated for at least 60 generations under the conditions with (+) andwithout (−) selection agent (250 nM methotrexate).

Data of the hCMV-MIE promoter methylation rate (mC-425), the copy numberper cell from (PSB) as well as the percentage alteration over 60generations of specific productivity (AqP) were determined. Recording ofdata was done as previously described by Osterlehner et al.

TABLE 4 Data of 12 project T cell lines. mC-425 is the mean ofpercentage methylation of cytosine at position 425 upstream oftranscription start site (TSS; −179 relative to TSS) of hCMV- MIE atbegin of cultivation phase (PSB); ΔqP is the percentage alteration ofspecific productivity (qP) over a time period of 60 cell generations;the average copy number of the light chain (LC) of the IgG transgene wasmeasured at begin of cultivation (PSB). Project T: 12 CHO antibodyproducing cell lines. Generated by transfection of CHO-K1-M with plasmidp5057 Sample mC-425 (%) ΔqP (60 generations) in % LC-Copies/Cell No. PSB+MTX −MTX PSB T-1 0.44 −35.40 −34.20 4.4 T-2 2.20 −30.60 −72.00 11.5 T-33.54 −81.60 −96.60 9.4 T-4 0.92 −23.40 −47.40 4.6 T-5 4.41 −15.60 −22.205.3 T-6 0.47 −14.40 −20.40 2.6 T-7 0.22 −16.20 −31.80 1.6 T-8 52.40−64.80 −79.20 64.3 T-9 52.84 −27.60 −49.20 67.5 T-10 49.61 3.60 1.2070.5 T-11 2.02 −10.20 −21.60 4.6 T-12 61.34 −32.40 −22.80 11.5

TABLE 5 Data of 20 project H cell lines. mC-425 is the mean ofpercentage methylation of cytosine at position 425 upstream of TSS ofhCMV-MIE at begin of cultivation phase (PSB); ΔqP is the percentagealteration of specific productivity (qP) over a time period of 60 cellgenerations; the average copy number of the light chain (LC) of the IgGtransgene was measured at begin of cultivation (PSB). Project H: 20 CHOantibody producing cell lines. Generated by transfection of CHO-K1-Mwith Plasmid p7672 Sample me of C-425 (%) ΔqP (60 generations) in %LC-Copies/Cell No. PSB +MSX −MSX PSB H-1 15.7 −46.80 −80.20 168.8 H-29.9 −71.00 −70.30 2.0 H-3 16.5 −73.10 −89.60 3.2 H-4 10.5 −69.30 −69.502.9 H-5 11.0 −40.40 −48.80 1.8 H-6 7.1 −79.50 −71.70 37.6 H-7 4.8 −66.20−81.50 53.0 H-8 4.4 −67.40 −75.70 30.6 H-9 23.4 −87.00 −77.00 95.1 H-1026.2 −16.40 −73.30 48.8 H-11 0.2 −43.10 −96.10 3.3 H-12 0.1 −42.00−33.00 1.5 H-13 1.3 8.00 −18.50 1.1 H-14 0.0 6.80 5.40 1.3 H-15 0.1−16.50 −40.40 3.5 H-16 0.4 16.20 −32.80 3.0 H-17 1.0 −13.20 −31.30 4.2H-18 46.3 −67.50 319.40 8.8 H-19 16.7 −11.20 2.70 10.1 H-20 21.8 −40.30−80.60 4.8

For investigation of the relative amount of histone modification viableCHO cells, which bear recombinant gene driven by the hCMV-MIE promoter,were harvested. After fixation in 3.7% formaldehyde lysed chromatin wassonicated and cross-linked DNA-histone complexes were purified accordingto appropriate histone modification. Subsequently the accumulatedDNA-histone complexes were degraded with proteinase K and DNA fragmentswere eluted. The amounts of various DNA fragments were compared withqPCR, using specific primer pairs.

To verify antibody-histone binding consistency with respect to thecorresponding modification of reference region, various genes weretested for accumulation of a specific histone modification. At first,the amount of antibody filtered histone modification close to thereference regions was compared to the purified sample of a non-specificno antibody control (mock) in project T and project H in one sampleeach. DNA fragments of antibody purified samples, mock and untreatedinput sample were used as templates for qPCR. Primer pairs of potentialreference regions were added to each sample and qPCR was performed intriplicates. The obtained Cq values were compared as percentage of theinput sample. In both projects, the antibody purified samples obtainedhigher values than mock in the appropriate region. This confirmed properbinding. Furthermore the activation marks H3ac and H3K4me3 accumulatedat the active regions Eif3i and Gusb. In contrast the silenced regionFox2a had higher concentration of H3K9me3 whereas H3K27me3 wasstrikingly located at the Gata5 region. The different histonemodifications matched to the corresponding control regions in bothprojects.

The estimation of relative amount of histone modification on the targetregion is strictly dependent on a stable modified reference region.

Normalization of a specific histone modification close to the hCMV-MIEpromoter to a reference sequence enables the comparison of differentcell lines. For this purpose the reference sequence has to have robustand reproducible levels of the respective histone modification.

In following table the average Cq values and the coefficient ofvariation (CV) of all cell lines, including the three biologicalreplicates are displayed for project T and project H.

TABLE 6 Reference regions in project T and H. The averages and theappropriate coefficient of variation (CV) of Cq values of all ChIPsamples including the biological triplicates were calculated for bothprojects; the rows display the different reference regions within thegenome; the columns contain the specific histone modifications; thechosen control regions for the appropriate epigenetic modifications aremarked in bold; therefor the Cq distance to mock and the CV weredecisive; in both projects, the activation marks H3ac, H3K4me3 as wellas total histone 3 are stable accumulated at the Gusb and the Eif3iregions (CV of 2%); the lower Cq values determines Gusb as reference foractivation marks; all potential reference regions had stable totalhistone 3 values. Project T: Cq values of ChIP Samples H3 H3ac H3K4me3H3K9me3 Target mean CV mean CV mean CV mean CV Eif3i 431 28.24 0.0127.57 0.02 25.45 0.02 34.35 0.03 Gusb 386 27.16 0.01 26.73 0.02 24.640.02 33.38 0.03 Fox2a 484 26.32 0.01 36.43 0.05 36.20 0.05 31.54 0.02Gata5 468 25.49 0.02 33.34 0.03 34.98 0.03 32.96 0.04 Rho 388 27.29 0.0235.56 0.02 36.66 0.03 35.17 0.03 Unc13c 474 26.81 0.01 35.40 0.03 36.350.05 32.01 0.02 Project H: Cq values of ChIP Samples H3 H3ac H3K4me3H3K9me3 Target mean CV mean CV mean CV mean CV Eif3i 431 26.41 0.0227.81 0.02 27.88 0.03 35.66 0.06 Gusb 386 25.98 0.02 27.65 0.03 27.850.03 34.96 0.06 Fox2a 484 25.96 0.02 34.98 0.06 35.45 0.07 34.69 0.06Gata5 468 25.75 0.02 33.61 0.05 35.30 0.07 35.16 0.07 Rho 388 27.08 0.0234.79 0.04 35.91 0.06 35.82 0.06 Unc13c 474 26.70 0.02 35.02 0.05 35.030.06 34.79 0.05 Project T: Cq values of ChIP Samples H3K27me3 Mock InputSample Target mean CV mean CV mean CV Eif3i 431 35.11 0.03 35.80 0.0322.38 0.01 Gusb 386 33.90 0.03 35.76 0.04 22.32 0.02 Fox2a 484 33.600.02 34.64 0.04 21.31 0.03 Gata5 468 30.70 0.03 34.61 0.04 21.72 0.01Rho 388 35.15 0.02 36.26 0.02 22.91 0.02 Unc13c 474 34.03 0.02 34.730.18 22.50 0.01 Project H: Cq values of ChIP Samples H3K27me3 Mock InputSample Target mean CV mean CV mean CV Eif3i 431 34.34 0.05 35.55 0.0622.94 0.02 Gusb 386 33.69 0.04 35.01 0.06 22.63 0.03 Fox2a 484 30.860.03 35.46 0.08 22.96 0.03 Gata5 468 30.09 0.03 34.88 0.06 22.62 0.03Rho 388 33.05 0.03 36.10 0.06 23.90 0.03 Unc13c 474 31.50 0.02 35.120.05 23.55 0.03

Gusb as the reference region for active histone modification H3ac andH3K4me3 and Gata5 as a stable region for H3K27me3 differ to theappropriate mock control and had consistent Cq values. Active controlregions Eif3i and Gusb are highly stable. For all control regions astable accumulation of total histone 3 (H3) was observed.

Biological triplicates of CHO cell lines recombinantly expressing anantibody project were analyzed for histone modification close to thelocation of the hCMV-MIE promoter. For this chromatin fragments werepurified using antibodies against specific histone modifications,digested to obtain protein free genomic DNA and quantified by real-timePCR (see FIG. 26 ). Normalization was executed for each used referenceregion. To determine the amplification efficiency of each primer pairthe LinReg PCR software, version 2014.5 was used (Ruijter, 2009,LinRegPCR). Amplification efficiencies of all primer-template pairs arequite similar. Therefore the Livak method was used for normalization,which assumes similar amplification efficiencies.

TABLE 7 Primer efficiencies. Amplification efficiencies were determinedfor all cell lines including biological triplicates; the primer pair(396/397) was used; for this purpose the qPCR raw data were applied tothe LinRegPCR software, version 2014.5 and efficiencies were calculated;rows are distinguished by the different primer pairs; the mean columncontains the average amplification efficiencies of each primer-templatepair and the Stdev column contains the appropriate standard deviation;distances between all amplification efficiencies are less than 2%; thus,the Livak method was used for normalization. Amplification efficiencywith LinReg PCR Project T Project H Primer pairs mean Stdev Primer pairsmean Stdev CMV 396/397 1.909 0.033 CMV 396/397 1.919 0.032 Gusb 386/3871.923 0.036 Gusb 386/387 1.935 0.037 Eif3i 431/432 1.893 0.036 Eif3i431/432 1.923 0.034 Fox2a 484/485 1.925 0.029 Fox2a 484/485 1.946 0.031Gata5 468/467 1.914 0.033 Gata5 468/467 1.934 0.034 Rho 388/389 1.8760.036 Rho 388/389 1.902 0.037 Unc13c 474/4745 1.904 0.032 Unc13c474/4745 1.927 0.034

The normalization by Livak comprises two normalization steps:

-   -   in the first step, the amount (Cq) of histone 3 modification        close to the hCMV-MIE promoter is normalized to the amount (Cq)        of histone 3 modification at the reference region→ΔCq    -   in the second step, the normalized histone 3 modification is set        to the normalized total histone 3 close to the hCMV-MIE        promoter→ΔΔCq

This finally displays the relative amount of modification per histone 3close to hCMV-MIE promoter.

Total histone 3 is stable in all reference regions allowing thenormalization of histone modification and total histone 3 with the samereference region. This minimizes variation within the normalizationprocess. For instance, the relative Histone acetylation per Histone 3was normalized to the control region Gusb, as the Gusb region comprisesa stable amount of the modified histone and the total Histone 3. Thefollowing formula was used for calculation:

$\left. {{\Delta\Delta}{Cq}}\rightarrow{ratio} \right. = \frac{2^{{\Delta{Cq}}_{H3\_{acetylation}}({{Cq}_{Gusb} - {Cq}_{CMV}})}}{2^{{\Delta{Cq}}_{H3\_{total}}({{Cq}_{Gusb} - {Cq}_{CMV}})}}$

Relative amounts of histone modification per histone 3 (ΔΔCq) and totalhistone 3 (ΔCq) close to hCMV-MIE promoter were plotted against the ΔqPvalues. Total histone 3 close to hCMV-MIE promoter were normalized toGusb and fitted against the ΔqP values. In addition histone 3 perrelative copy number (H3/rCN) and mock per histone 3 (Mock/H3) wereexamined. H3/rCN subtracts ideally the copy number and is a measurementfor the relative histone 3 density (H3D) close to hCMV-MIE promoter.Mock/H3 is the unspecific effect of background noise per histone 3.

Correlations were examined with a standard least squares fit model. Tothis end the software JMP10 (JMP®10.0.1 Release: 2, 64-bit edition; SASInstitute Inc.) was used.

TABLE 8 Measurement of effect of epigenetic modifications on long termproduction stability. P-values of effects on ΔqP with (+) and without(−) selection pressure (250 nM MTX) are displayed in the columns; alleffects are measured close to hCMV-MIE promoter and ordered in rows;effects of normalized histone 3 (H3), density of histone 3 per relativecopy number (H3/rCN), background noise per histone 3 (Mock/H3) andmodifications per histone 3 (H3ac/H3, H3K4me3/H3, H3K27me3/H3 andH3K9me3/H3) were calculated in a standard least square model withsoftware JMP10; in project H, acetylation per histone 3 (H3ac/H3) has ahighly significant effect on ΔqP if cells were cultivated underselection pressure (+), also H3/rCN, Mock/H3, H3K4me3, H3K27me3 and H3events resulted in moderate to high significant effects under ΔqP+condition. Single effect leverage model: P-values for an epigeneticresulted effect Project T Project H Epigenetic marks ΔqP+ ΔqP− ΔqP+ ΔqP−H3 0.116 0.172 0.0013 0.036 H3/rCN 0.7981 0.5291 0.2273 0.8701 Mock/H30.5319 0.4735 0.0286 0.9214 H3ac/H3 0.378 0.6866 <0.0001 0.1449H3K4me3/H3 0.637 0.8012 0.0102 0.8896 H3K27me3/H3 0.4676 0.8338 0.0050.5864 H3K9me3/H3 0.4771 0.6858 n.d. n.d. = not determined

The degree of acetylation per histone 3 (H3ac/H3) at the hCMV-MIEpromoter has a highly significant effect on the loss of productivity,obtained over 60 generations at the presence of selection agent MTX.

Further significant effects (listed from weakest to strongest effect)were observed for H3/rCN, Mock/H3, H3K4me3/H3, H3K27me3/H3 and H3. Inconsideration of the effect levels for Mock/H3 and H3 alone H3 alone hasa dominant influence on the combined effect X/H3. Epigenetic events withgreater effects than H3 alone have been found to be a good predictionmarker:→H3ac/H3.

A Jackknife outlier analysis was performed. Thereby one outlier intriplicate was found by comparing ΔqP of both selection agentconditions. In regard to the analysis, cell line H-18 was excluded fromeffect measurements. Thereafter the samples of project H were analyzedagain with the effect screening model after exclusion of cell line H-18.It has been found that the loss of acetylation per histone 3 (H3ac/H3)at the hCMV-MIE promoter has the most significant effect on the loss ofproductivity, obtained over 60 generations under both selectionconditions.

TABLE 9 Effect measurements of modifications on long term stability inproject H after exclusion of outlier cell line H-18. P-values of effectson ΔqP with (+) and without (−) selection pressure (250 nM MTX) aredisplayed in the columns; all effects are measured close to hCMV-MIEpromoter and ordered in rows; effects of normalized histone 3 (H3),density of histone 3 per relative copy number (H3/rCN), background noiseper histone 3 (Mock/H3) and modifications per histone 3 (H3ac/H3,H3K4me3/H3, H3K27me3/H3 and H3K9me3/H3) were calculated in a standardleast square model with software JMP10; most significant effect on ΔqPcould be observed for H3ac/H3 under both selection conditions. Singleeffect leverage model of project H: P-values for an epigenetic resultedeffect Epigenetic marks ΔqP+ ΔqP− H3 0.0005 0.0003 H3/rCN 0.2600 0.3016Mock/H3 0.0475 0.0485 H3ac/H3 <0.0001 0.0001 H3K4me3/H3 0.0138 0.3464H3K27me3/H3 0.0072 0.0078

In project T the plot of H3ac/H3 against ΔqP shows a simultaneousincrease of long term stability and the acetylation per histone 3,particularly under selection pressure.

To account for different levels of histone 3 at the hCMV-MIE promoterhistone 3 acetylation was further normalized to histone 3 (H3ac/H3).

Relative histone 3 acetylation levels (H3ac/H3) were compared withalterations in specific productivity (ΔqP) over 60 generations in thepresence (+) and in the absence (−) of selection agent MSX. Afteroutlier analysis, the standard least squares regression model was fedwith H3ac/H3 values of biological replicates and the ΔqP values of 19CHO cell lines. Significant correlation of H3ac/H3 and ΔqP was detected.

For H3ac/H3 values a decision tree was calculated with jmp software.Best split node and therefore best filter was calculated with theLogWorth statistic. The best split under (+) MSX condition wascalculated to be 0.58 histone 3 acetylation relative to the level ofHistone 3 (H3ac/H3) for the samples H. In order to reduce the number offalse negative Samples the filter was set to the lower value of A>0.5H3ac/H3.

TABLE 10 The means of delta SPR (ΔqP) at conditions with or withoutselection agent MTX or MSX, respectively, were calculated for eachpositive gate and compared to the unfiltered mean and is presentedbelow: samples H-1 to H-17, Mean ΔSPR Mean ΔSPR H-19, H-20 N (+) MSX(−)MSX unfiltered 56 −0.39 −0.55 A >0.5 H3ac/H3 23 (41%) −0.15 −0.38Mean ΔSPR Mean ΔSPR samples T-1 to T-12 N (+) MTX (−) MTX unfiltered 36−0.29 −0.41 A >0.5 H3ac/H3 7 (19%) −0.19 −0.32

The filter A>0.5 H3ac/H3 increases the mean delta SPR compared to theunfiltered condition.

B.3. Combination of Promoter Methylation and Histone Acetylation

Relative levels of histone 3 acetylation relative to the level ofhistone 3 close to human CMV major-immediate-early promoter/enhancerfragment and percentage of C-425 methylation of human CMVmajor-immediate-early promoter/enhancer fragment were investigated asprediction markers for production stability with and without selectionagent.

TABLE 11 The means of delta at conditions with or without selectionagent were calculated for each positive gate and compared to theunfiltered mean and is presented below: samples H-1 to H-17, Mean ΔSPRMean ΔSPR H-19, H-20 N (+) MSX (−)MSX unfiltered 56 −0.39 −0.55 A >0.5H3ac/H3 23 (41%) −0.15 −0.38 B <5% mC-425[%] PSB 27 (48%) −0.24 −0.45Combined filter (B ∩ A) 20 (36%) −0.13 −0.36 Mean ΔSPR Mean ΔSPR samplesT-1 to T-12 N (+) MTX (−) MTX unfiltered 36 −0.29 −0.41 A >0.5 H3ac/H3 7(19%) −0.19 −0.32 B <5% mC-425[%] PSB 24 (67%)  −0.28 −0.43 Combinedfilter (B ∩ A) 7 (19%) −0.19 −0.32

The combination of filter A>0.5 H3ac/H3 with filter B<5% mC-425[%] PSBincreases the mean delta SPR compared to the unfiltered condition.

In more detail a decision tree was calculated using the Jmp 10 softwareto determine the threshold value of the H3ac/H3 ratio in order to allowexclusion of bad producers. The LogWorth statistic was used to identifythe best split node. The LogWorth is calculated as: −log 10 (p-value),where the adjusted p-value is calculated in a complex manner that takesinto account the myriad number of different ways splits can occur (Sall,2002, Monte Carlo Calibration of Distributions of Partition Statistics;SAS white paper.). The best split for ΔqP+ condition was 0.47acetylation per histone 3.

To minimize false positive values the threshold value was set to 0.5H3ac/H3 and transferred to both conditions in projects H and T.

In addition, the threshold of 5% for hCMV-MIE promoter methylation (%mC-425) was determined from the recorded methylation data (see Tablesabove) to use the (synergistic) combination of both prediction markers.Thus, samples with more than 5% DNA methylation or less than 0.5acetylation per histone 3 at the hCMV-MIE promoter were excluded.

The average of ΔqP was calculated for positive filtered samples andcompared with the average ΔqP of the unfiltered samples. This was doneunder consideration of whether or not MTX was added to the culturemedium.

The use of the prediction marker H3ac/H3 results in an increase of longterm stability of included samples.

The average ΔqPs after filtration were almost identical in bothprojects.

TABLE 12 Exclusion of bad producers. Samples with values above 0.5H3ac/H3 and below 5% mC-425 were used to calculate the average ΔqP;interestingly filtered ΔqPs were almost identical for both projects. NMean ΔqP+ Mean ΔqP− Project T unfiltered 36 −0.29 −0.41 A >0.5 H3ac/H3 7 (19%) −0.19 −0.32 B <5% mC-425 24 (67%) −0.28 −0.43 Project Hunfiltered 56 −0.39 −0.55 A >0.5 H3ac/H3 23 (41%) −0.15 −0.38 B <5%mC-425 27 (48%) −0.24 −0.45

The degree of histone 3 acetylation at the hCMV-MIE promoter had asignificant effect on stability. Also the amount of total H3 and degreeof methylation at position C-425 (% mC-425) had effects on the long termstability. Furthermore the found negative correlation of H3ac/H3 and %mC-425 confirms the reliability of these markers.

The threshold setting leads to the exclusion of all bad producers. Cellsabove this value are predominately stable.

B.4. Combination of Promoter Methylation and Histone Acetylation andTransgene Copy Number

The correlation of histone 3 acetylation with the DNA methylation andthe copy number were significant, which provides evidence for theinterrelation of the copy number, the DNA methylation and the histone 3acetylation. It has been found that the DNA methylation degree wascontrary to the histone 3 acetylation. It has been further found thatcopy number versus histone 3 acetylation and DNA methylation providesfor the fact that cell lines with a higher copy number are mostlynon-acetylated and prone for DNA methylation. It has been found thecorrelations of copy number and the alteration of long-term productivityin the presence and absence of selection agent. Thus, the copy numberinteracts with the degree of marks, which in turn influence thelong-term production stability.

In addition an unintended accumulation of instable cell lines ispromoted in the standard cell line development by the early selectioncell lines according to their antibody titers. It is assumed that celllines with a low copy number need to be predominately active whereasproducers with a high copy number can have a plethora of differentepigenetic statutes from non-over moderate- to high-activation of eachindividual transgene as long as the sum of the transcripts arecomparable. Considering this, the first selection according to theantibody titer either prefers cell lines with high copy numbers whichmight have stable integration sites by coincidence or low copy numbercell lines which are slightly forced to have them. The copy number frombegin of cultivation phase was plotted against the stability and it wasfound that cell lines with high number of transgenes were prone forinstability.

Thus, herein is reported as one aspect a three step cell line selectionmethod/process. In the first step antibody expressing cells with a lowlight chain copy number are expanded, in the second step a selectionaccording to their histone 3 acetylation degrees is performed.Thereafter a fast reduction of the selection agent is performed. Therebycell lines are obtained with stable integration sites, a reduced riskfor gene silencing and for copy number loss as well as theidentification of subpopulations with pre-stressed destabilizingmechanisms.

In one embodiment the copy number of stably integrated light chainexpression cassettes is below 50. In another embodiment the copy numberof stably integrated light chain expression cassettes is below 25. In afurther embodiment the copy number of stably integrated light chainexpression cassettes is below 10. In a further embodiment the copynumber of stably integrated light chain expression cassettes is below 6.

The following examples, sequence listing and figures are provided to aidthe understanding of the present invention, the true scope of which isset forth in the appended claims. It is understood that modificationscan be made in the procedures set forth without departing from thespirit of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Number of methylated CpG site of the hCMV-MIE promoter/enhancerobtained from different cell lines.

FIG. 2 Plasmid map of p5057.

FIG. 3 A: Specific production rate in the absence of a selection agentof cell line K18.1 over multiple generations in a long-term production.

-   -   B: Specific production rate in the absence of a selection agent        of cell line G25-10 over multiple generations in a long-term        production.    -   C: Specific production rate in the absence of a selection agent        of cell line G25-17 over multiple generations in a long-term        production.    -   D: Specific production rate in the absence of a selection agent        of cell line G42-5 over multiple generations in a long-term        production.    -   E: Specific production rate in the absence of a selection agent        of cell line 43-16 A10 over multiple generations in a long-term        production.

FIG. 4 Upper figure: frequency of methylation within hCMV-MIE DNApromoter/enhancer from recombinant CHO cell lines at differentmethylation sites determined by the analysis of 19-22 individualpromoter nucleic acids; lower figure: schematic representation ofmethylated CpG sites within hCMV-MIE promoter/enhancer DNA fromrecombinant CHO cell lines—methylated sites are shown in black—position425 is highlighted by an arrow.

-   -   A: cell line K18.1—methylation of all CpG sites: 12%,        methylation of site C425: 64%, methylation of site C591: 27%,        methylation of site C96: 32%;    -   B: cell line G25-10—methylation of all CpG sites: 0.5%,        methylation of site C425: 0%, methylation of site C591: 5%,        methylation of site C96: 0%;    -   C: cell line G25-17—methylation of all CpG sites: 0.3%,        methylation of site C425: 0%, methylation of site C591: 0%,        methylation of site C96: 0%;    -   D: cell line G42-5—methylation of all CpG sites: 0.6%,        methylation of site C425: 0%, methylation of site C591: 0%,        methylation of site C96: 0%;    -   E: cell line 43-16 A10—methylation of all CpG sites: 4.4%,        methylation of site C425: 25%, methylation of site C591: 15%,        methylation of site C96: 10%.

FIG. 5 Discrimination of hCMV-MIE promoter/enhancer which is methylatedat site 425 from hCMV-MIE which is non-methylated at site 425 bymethylation specific real-time qPCR; PCR amplification curves fortemplates #11 (A), #62 (B), #01 (C) and #04 (D).

FIG. 6 PCR amplification curves for different methylation specificprimer and primer pairs.

FIG. 7 Recovery of site 425 methylated hCMV-MIE promoter/enhancer in thebackground of non-methylated hCMV-MIE promoter/enhancer by methylationspecific real-time qPCR.

FIG. 8 hCMV-MIE promoter/enhancer nucleic acid methylation atmethylation site 425 obtained with primer #239 and #267 withpre-amplification (A) and directly from bisulfite treated genomic DNA(B).

FIG. 9 hCMV-MIE promoter/enhancer nucleic acid methylation atmethylation site 425 obtained with primer (black) 239+237 and 239+267,(white) 263+237 and 263+267, (horizontal lines) 264+237 and 264+267 and(vertical lines) 239+237 and 239+266.

FIG. 10 Exemplary high resolution normalized melting curve analysis (A)and first derivative thereof, i.e. melting peaks (B).

FIG. 11 Correlation of the degree of methylation at C425 beforelong-term cultivation and the relative alteration of the SPR after 60generations cultivation with 250 nM MTX (A) or without MTX (B).

FIG. 12 Correlation of the degree of methylation at C425 beforelong-term cultivation and the degree of methylation after 60 generationscultivation with 250 nM MTX.

FIG. 13 Schematic representation of methylated CpG sites within hCMV-MIEpromoter/enhancer DNA from clone 44-28. Methylated sites are shown inblack. Nucleotide position 425 is highlighted by an arrow. Methylationof all CpG sites: 18%; methylation at C425: 80%; methylation at C591:70%; methylation at C96: 60%.

FIG. 14 Light chain gene copy numbers before and after stability testingwithout MTX.

FIG. 15 Relative Levels of histone 3 acetylation relative to the levelof histone 3 close to human CMV major-immediate-early promoter/enhancerfragment normalized to reference gene Gusb.

The variances between the 3 biological replicates are displayed by thestandard deviation.

FIG. 16 Relative Levels of histone 3 Lysine 4 three-fold methylationrelative to the level of histone 3 close to human CMVmajor-immediate-early promoter/enhancer fragment normalized to referencegene Gusb. The variances between the 3 biological replicates aredisplayed by the standard deviation.

FIG. 17 Actual by Predicted Plot of effect H3K4me3/H3 for samples H-1 toH-20 with 3 biological replicates. Confidence curves for the line of fitare shown in dashed lines to provide visual indication of whether thetest of interest is significant at the 5% level. The curves need tocross the horizontal line for significance.

FIG. 18 Actual by Predicted Plot of effect H3ac/H3 for the samples H-1to H-20 with 3 biological replicates. Confidence curves for the line offit are shown in dashed lines to provide visual indication of whetherthe test of interest is significant at the 5% level. The curves need tocross the horizontal line for significance.

FIG. 19 Actual by Predicted Plot of effect mC 425[%] for the samples H-1to H-20. Confidence curves for the line of fit are shown in dashed linesto provide visual indication of whether the test of interest issignificant at the 5% level. The curves need to cross the horizontalline for significance.

FIG. 20 Outlier analysis of project H with Jackknife Distances for deltaSPR with and without MSX as condition. Sample H-18 is far beyond theUpper Control Limit (UCL), which strongly indicates that sample as anoutlier.

FIG. 21 Actual by Predicted Plot of effect H3K4me3/H3 for samples H-1 toH-17, H-19 and H-20 with 3 biological replicates. Confidence curves forthe line of fit are shown in dashed lines to provide visual indicationof whether the test of interest is significant at the 5% level. Thecurves need to cross the horizontal line for significance.

FIG. 22 Actual by Predicted Plot of effect H3ac/H3 for samples H-1 toH-17, H-19 and H-20 with 3 biological replicates. Confidence curves forthe line of fit are shown in dashed lines to provide visual indicationof whether the test of interest is significant at the 5% level. Thecurves need to cross the horizontal line for significance.

FIG. 23 Actual by Predicted Plot of effect mC-425[%] for samples H-1 toH-17, H-19 and H-20. Confidence curves for the line of fit are shown indashed lines to provide visual indication of whether the test ofinterest is significant at the 5% level. The curves need to cross thehorizontal line for significance.

FIG. 24 Delta SPR values of samples H-1 to H-17, H-19 and H-20 plottedin histograms. Different Selection and filter conditions are displayed.The horizontal line within the box represents the median. The confidencediamond contains the mean, the upper and lower 95% of the mean. Themiddle of the diamond represents the mean. The top and bottom points ofthe diamond represent the 1^(st) and 3^(rd) quartiles. The box has linesthat extend from each end, called whiskers. The whiskers extend from theends of the box to the outermost data point that falls within thedistances computed as 1^(st) quartile −1.5*(interquartile range) and3^(rd) quartile+1.5*(interquartile range). If the data points do notreach the computed ranges, then the whiskers are determined by the upperand lower data point values (not including outliers). The bracketoutside of the box identifies the shortest half, which is the densest50% of the observations (Rousseuw and Leroy 1987).

FIG. 25 Delta SPR values of project T plotted in histograms. Differentselection and filter conditions are displayed.

FIG. 26 Scheme for chromatin immunoprecipitation (ChIP) of CHO cell lineDNA.

FIGS. 27A-F Correlation studies of epigenetic marks, copy number andstability. A: Plot of C-425 methylation to the acetylation per histone 3at the hCMV-MIE promoter. B: plot of the alteration of specificproductivity over 60 generations of CHO cells at the absence (−) andpresence (+) of selection agent MSX after exclusion of cell line H-18.High correlative behavior of stability of cell lines with and withoutselection agent was determined. C: Plot shows the correlation of copynumber and histone 3 acetylation, indicating that only cell lines withlow copy number were acetylated at histone 3. D: Plot displays thecorrelation of copy number and DNA methylation whereas only cell lineswith high copy number are predominantly methylated at the hCMV-MIEpromoter. E and F: plots show the correlation of copy number and thealteration of productivity over 60 generations in the presence (+) andabsence (−) of selection agent. Therefore high copy number cell linesare prone for silencing.

FIG. 28 Human antibody of class IgG expressing plasmid. Light and heavychain expression cassette of human immunoglobulin were both under thecontrol of a human CMV major immediate-early promoter and enhancer (SEQID NO: 01) (hCMV-MIE).

EXAMPLE 1

General Techniques

Solutions CHIP Cell lysis buffer 20 mM Tris-HCl pH 8.0 85 mM KCl 0.5%NP40 (Nonident P-40/octylphenolpolyethoxyethanol) double distilled H2ONuclei Lysis buffer 50 mM Tris-HCl pH 8.0 10 mM EDTA pH 8.0 1% SDSdouble distilled H2O 1 tablet Roche Complete per 10 ml Low salt washbuffer 0.1% SDS 1% Triton X 100 2 mM EDTA 20 mM Tris-HCl pH 8.0 150 mMNaCl double distilled H2O High salt wash buffer 0.1% SDS 1% Triton X 1002 mM EDTA 20 mM Tris-HCl pH 8.0 500 mM NaCl TE buffer 10 mM Tris-HCl pH8.0 1 mM EDTA (IP) Elution buffer 50 mM NaHCO3 1% SDS double distilledH2O IP Dilution buffer 0.01% SDS 1.1% Triton X 100 1.2 mM EDTA 16.7 mMTris-HCl pH 8.1 (8.0) 167 mM NaCl H2O IP Blocking Buffer IP Dilutionbuffer BSA Salmon Sperm DNA Roche Complete Protease Inhibitor

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook etal., Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). The molecularbiological reagents were used according to the manufacturer'sinstructions.

DNA Sequence Determination

DNA sequencing was performed at SequiServe GmbH (Vaterstetten, Germany).

DNA and Protein Sequence Analysis and Sequence Data Management

The EMBOSS (European Molecular Biology Open Software Suite) softwarepackage and Invitrogen's Vector NTI version 9.1 were used for sequencecreation, mapping, analysis, annotation and illustration.

Protein Determination

A chromatographic method was used to quantify the amount of antibodypresent in a sample. A Poros A column was used that binds the Fc-regionof the antibody. The antibody binds to the column and is subsequentlyeluted by low pH conditions. Protein concentration was determined bydetermining the optical density (OD) at 280 nm, with a referencewavelength of 320 nm, using the molar extinction coefficient calculatedon the basis of the amino acid sequence.

Agarose Gel Electrophoresis

Agarose gel electrophoresis was performed to analyze the quality, sizeand amount of linear DNA fragments (Chong, 2001). According to the sizeof DNA fragments, agarose solutions of 1% (w/v) were dissolved in 1×TAEsolution by boiling. Gels contained a final concentration of 0.5 μg/mlethidium bromide. Samples were prepared by adding 1/6 (v/v) of 6×DNAloading dye. A DNA ladder was used as a size standard. Electrophoresiswas performed in 1×TAE by applying 10 V/cm gel length (not more than 250V). After separation, DNA was examined under UV light (254-366 nm) in agel documentation system (Intas Science Imaging Instruments GmbH,Goettingen, Germany).

Gel Extraction

After Agarose Gel Electrophoresis, the gel slice with the favored bandwas excised by scalpel and the gel slice was further extracted byQIAquick® Gel Extraction Kit (Qiagen, Hilden, Germany). Following this,3 volumes of Buffer QG were added to 1 volume of gel (100 mg˜100 μl) andincubated for 10 minutes at 50° C. One gel volume of isopropanol wasadded to the dissolved gel and mixed. DNA fragments adhered to QIAquickspin column by one minute centrifugation at 13,000 rpm and were washedby 0.75 ml Buffer PE and subsequent centrifugation (13,000 rpm, 1 min.).Second wash in another collection tube removed the residual wash buffer.Finally DNA was eluted in 30 μl RNAse-free H₂O into a sample tube bycentrifugation (13,000 rpm, 1 min.). DNA can be checked on Flash-gel(FlashGel™ systems, Lonza, Cologne, Germany).

DNA Quantification

Nanodrop 2000 (PEQLAB Biotechnologie GmbH, Erlangen, Germany) was usedto quantify DNA concentration by measuring the optical density (OD) at awavelength of 260 nm. The purity of the DNA can be judged by the ratioOD 260/280 and 260/230. Pure DNA preparations should possess a ratio≥1.8 and 2.0.

Transformation of Competent Bacteria

Forty-five microliter of chemically competent E. coli NEB 5 alpha (NEB,Germany) were thawed on ice for 10 minutes and thereafter incubated with1 pg-100 ng of plasmid DNA (1-5 μl) for 30 minutes on ice. The cellsuspension was heat shocked at 42° C. for 45 seconds and immediatelychilled on ice for 5 minutes. Nine hundred and fifty microliter of roomtempered SOC or LB was added and suspension was incubated at 37° C. for60 minutes with agitation (250 rpm). Transformed bacteria were plated onpre-warmed agar plates containing ampicillin. Plates were incubatedovernight at 37° C.

Plasmid Preparation

Plasmids were prepared using the Qiagen Plasmid Mini and Maxi Kits(Qiagen, Hilden, Germany) following the manufacturer's instructions.

DNA Purification

Genomic DNA was isolated and purified with the Allprep DNA/RNA Mini Kit(50) (Cat. No. 80204, Qiagen, Hilden, Germany) and the DNAse Blood &Tissue Kit (Qiagen, Hilden, Germany) according to manufacturer'srecommendations. Purification of fragments was done with the High PurePCR Product Purification Kit (Roche Diagnostics GmbH, Mannheim, Germany)by following the manufacturer's instructions.

Phenol-Chloroform Extraction

Plasmid concentration and purification was done by phenol/chloroformextraction. All steps were performed under a chemical hood. Five hundredmicroliter of linearized plasmid was mixed with one volume Roti®Phenol/chloroform/isoamylalcohol (25:24:1) (Cat. No. A156, Roth,Germany) and vortexed for 30 seconds. Thereafter the emulsion wastransferred in pre-centrifuged Phase Lock Gel™ Light tube (Cat. No.0032005.101, Eppendorf, Hamburg, Germany) for centrifugation (13,000rpm, 1 minute). Upper aqueous phase was transferred into new tube andthe extraction was repeated. Thereafter the upper aqueous phase wasmixed with 500 μl chloroform/isoamylalcohol (24:1) and extraction stepswere repeated again once. The upper phase was transferred into a newtube and mixed with 0.1 start volume of 3 mol/L sodium acetate buffer(pH 4.8-5.2) and 0.7 start volume of 100% isopropanol (20-30° C.) byinverting the tube 4-6 times. After 20 minutes incubation at −80° C.followed by centrifugation (13,000 rpm, 30 minutes at 4° C.) thesupernatant was discarded. The pellet was washed with chilled 1 ml 70%ethanol, centrifuged (13,000 rpm, 5 minutes at 4° C.) and thesupernatant was discarded. The wash step was repeated under sterileconditions and residual supernatant was completely removed. The pelletwas dried for 5-10 minutes on sterile air and subsequently taken up in0.5 start volume double distilled water.

Transgene Copy Number Determination

Determinations of transgene copy number as well as methylation rate ofC-425 of hCMV-MIE promoter were performed according to therecommendations of Osterlehner et al. (supra).

Relative Copy number (rCN) was calculated by two to the power of thedifference of the Cq value of the reference input sample (e.g. Gusb)from the Cq value of the target input sample hCMV-MIE promoter.

Chromatin Immunoprecipitation

CHO cells lines were harvested with a viability of greater than 90%,preferably greater than 97%. Before fixation, beads for pre-clearing andprecipitation were prepared. Pre-clearing beads were generated byincubation (1 hour, RT while rotation) of 15 μl protein A agarose slurry(Roche Diagnostics GmbH, Mannheim, Germany) with 2 μl purified rabbitIgG (Cat. No. PP64B, Millipore, Germany) in 120 μl ChIP Dilution Bufferper sample. Two wash steps with each one volume of ChIP dilution bufferwere done for purification followed by resuspension of beads in 85 μlChIP dilution buffer. Fifteen microliter agarose A beads forprecipitation were blocked in 1 ml ChIP dilution buffer with 5 mg/ml BSA(Roche Diagnostics GmbH, Mannheim, Germany) and 100 μg/ml of preheatedsalmon sperm DNA (10 minutes at 95° C. and 5 minutes on ice; Cat No.15632-011, Invitrogen, Germany). Resuspended pellet were incubated for4-5 hours at 4° C. while rotation, subsequently washed three times andmounted in 2 volumes of ChIP Dilution Buffer.

About 1*10⁷ cells per sample were fixed by adding formaldehyde intomedia up to a final concentration of 3.7% and incubated for 10 minutesat RT as described previously (Beneke, S., et al. PLoS One 7 (2012)e32914). Fixation was stopped by adding glycine up to a one foldsolution. After two wash steps with ice cold PBS, and centrifugation at2000×g for 3 minutes, the pellet was resuspended in 1 ml PBS containingprotease inhibitor Roche complete (Roche Diagnostics GmbH, Mannheim,Germany). The suspension was pelleted by centrifugation (3000×g, 5minutes, 4° C.). The supernatant was discarded.

Lysis of the pellet was done by adding 1 ml cell lysis buffer plus Rochecomplete (Roche Diagnostics GmbH, Mannheim, Germany) and incubation onice for 10 minutes. After centrifugation (2300×g, 4 minutes, 4° C.) thenuclei pellet was resuspended in 300 μl Nuclei Lysis Solution andsonicated (Output 5, Duty cycle 90%, 15 seconds sonication followed by2-3 minutes incubation on ice for 6 cycles; Branson Sonifier B15,Dietzenbach, Germany). Seven hundred microliter of nuclei lysis solutionwere added to sonicated nuclei followed by centrifugation (13,000 rpm,15 minutes, 4° C.). The supernatant was transferred into a new tube forstorage at −80° C. until use. Sonification grade was tested afterprotein digestion on an agarose gel.

Chromatin was pre-cleared with 80 μl prepared protein A agarose slurryby incubation (2 hours at 4° C. while rotation) and centrifugation(13,000 rpm, 6 minutes, 4° C., Eppendorf, Hamburg, Germany). Supernatantwas transferred into new tube and protein concentration was determined(Pierce® BCA Protein Assay Kit, Thermo Scientific, Rockford, USA).Twenty-five to one hundred microgram of chromatin perimmunoprecipitation (IP) were mounted in 200 μl nuclei lysis buffer andadded to 300 μl ChIP dilution buffer comprising 3 μg of appropriateantibody. Immunoprecipitation was done overnight at 4° C. while underrotation. Undiluted input samples were stored at −20° C. After overnightincubation, 40 μl of blocked protein A agarose beads were added to IPsolution and incubated (1 hour at 4° C. while rotating). The precipitatewas washed with twice with low-, once with high-salt wash buffer, oncewith LiCl wash buffer and once with TE wash buffer. Each wash was donein 1 ml buffer for 5 minutes on rotating platform followed bycentrifugation (500×g, 30 seconds, 4° C.). After the last wash stepbeads were centrifuged with 500×g for 1 minute. Bead pellets werecombined with 200 μl IP elution buffer, while simultaneously 25-100 μgchromatin input sample were filled up to 200 μl with IP elution bufferand incubated for 30 minutes at 65° C. while shaking. Subsequently tubeswere incubated for 30 minutes at 37° C. after adding 0.5 μl RNAse DNAsefree (Roche Diagnostics GmbH, Mannheim, Germany) to each tube. For finalprotein digestion 10 μl 4 M NaCl (final conc. 0.2 M) and 2 μl proteinaseK (final conc. 100-200 μg/ml, Roche Diagnostics GmbH, Mannheim, Germany)were added to the reaction samples and incubated for 1.5 hours at 65° C.DNA was purified by Roche PCR purification kit (Roche Diagnostics GmbH,Mannheim, Germany).

Bisulfite Conversion

For conversion of non-methylated cytosines into uracil the EZ-96 DNAMethylation-Lightning™ kit (deep-well format) (ZymoResarch, Freiburg,Germany) was used according to manufacturer's instructions. In brief,gDNA was extracted with DNAse blood & tissue kit (Qiagen, Hilden,Germany) and concentration was measured with Nanodrop 2000 (PEQLABBiotechnologie GmbH, Erlangen, Germany) to set concentration at 350 nggDNA in 20 μl double distilled water. Twenty microliter gDNA were mixedwith 130 μl of lightning conversion reagent in a conversion plate. Theplates were incubated at 98° C. for 8 minutes, then at 54° C. for 60minutes and finally temporarily stored at 4° C. A Zymo-Spin™ I-96binding plate containing 600 μl of M-binding buffer per well was mountedon a collection plate. Samples from the conversion plate were added tothe Zymo-Spin™ I-96 binding plate, mixed and centrifuged (3000×g for 5minutes). Plates were washed with 400 μl of M-wash buffer andcentrifuged (3000×g, 5 minutes). 200 μl of L-desulphonation buffer wereadded to each well, incubated (20-30° C., 20 minutes) and subsequentlycentrifuged (3000×g for 5 minutes). An additional wash step wascompleted with 10 minutes centrifugation at 3000×g and converted DNA waseluted in 30 μl M-elution buffer. Converted DNA was stored at −20° C.until use.

Amplification of Converted and Integrated hCMV-MIE Promoter

TABLE 13 For investigation of CpG methylation ofhCMV-MIE promoter, primer pairs fordistal (F1 & R1) and proximal (F2 & R2)promoter region were designed as follows: UG_730_Bisul_GATATTGATTATTGATTAGTT SEQ ID CMV1 ATTAATAGTAATTAA NO: 44 F1UG_731_Bisul_ CAAATAAAAAAATCCCATAA SEQ ID CMV1 AATCATATACTAA NO: 45 R1UG_732_Bisul_ TTAGTATATGATTTTATGGGA SEQ ID CMV2 TTTTTTTATTTG NO: 46 F2UG_733_Bisul_ TTCTAATACTAAACTCCTCT SEQ ID CMV2 CCCAA NO: 47 R2

TABLE 14 Ten picomole of each primer (0.5 μl) were added to 12.5 μlZymoTaq ™ DNA polymerase premix (ZymoResarch, Germany), 3 μl gDNAtemplate and filled up to 25 μl with double distilled water. Polymerasechain reaction (PCR) was done with Mastercyler nexus X1 (Eppendorf,Hamburg, Germany), PCR conditions were as follows: 95° C. 10 min 94° C.30 sec    2 cycles 49° C. 2 min 68° C. 2 min 94° C. 30 sec 34-43 cycles54° C. 2 min 68° C. 2 min 72° C. 10 min  4° C. œ

Amplicons were purified with Roche PCR purification kit (RocheDiagnostics GmbH, Mannheim, Germany) in 30 to 40 μl Elution buffer.Concentration was defined with Nanodrop 2000 (PEQLAB BiotechnologieGmbH, Erlangen, Germany) and size of amplicons was displayed by agarosegel electrophoresis.

Inverse PCR

The inverse polymerase chain reaction (iPCR) is a method to determineintegration sites of a randomly integrated vector. From this the knownvector-sequence near the cutting site will be used to generate primersof at least 20 bp length. Those primers are oriented in oppositedirections.

The genome was digested with frequently cutting enzymes (4 bp-cutter)which did not cut between the primer binding sites and at thelinearization site of the vector. The resulting fragments are ligated atvery dilute conditions so as to favor intramolecular ligation. Thecircularized sequence can now be amplified with the inverse primers.

Genomic DNA was isolated with Qiagen blood & cell culture DNA prep. midikit (Qiagen, Hilden, Germany) out of 2×10⁷ cells (˜90 μg DNA) percondition (genomic DNA can be stored at −20° C. for several months) anddissolved in 90 μl double distilled water.

The isolated genomic DNA was digested with differing restrictionendonucleases at the appropriate working temperature for a minimum of 16hours. For the digestion of gDNA the Enzymes CviQI, MseI and MspI forthe iPCR upstream of PvuI integration site and the enzymes MseI, BfaIand MluCl for the iPCR downstream of the PvuI integration site wereused. Ten microgram genomic DNA were used per digestion (3.8×10⁶copies).

TABLE 15 Digestion: Component Volume/80 μl Final amount gDNA template xμl 10 μg NEBuffer 10× 8 μl BSA 10× (if required) 8 μl double distilledwater Add to 80 μl Restriction endonuclease x μl 50 U*

The digested DNA was purified with the Roche PCR-purification kit andeluted in x μl (200 μl) Elution buffer. The high dilution grade of DNAfavors intramolecular ligation.

TABLE 16 Ligation: Neb T4 DNA Ligase (M0202S) for iPCR Digested DNA 20μl (1 μg) (3.8 × 10⁵ copies) Ligation buffer, 10x  50 μl T4 DNA ligase(400 U*μl⁻¹)  1 μ1 WFI (double distilled water) 429 μl

In order to generate circular DNA fragments, the digested DNA wasligated overnight at 16° C. using T4 DNA ligase (NEB, Frankfurt/Main,Germany). Ligated DNA was eluted in 50 μl Elution buffer of the highpure PCR purification kit (Roche Diagnostics GmbH, Mannheim, Germany)and added to 10 ng template DNA. As a control group, non-transfectedgDNA for CHO cell line was investigated.

TABLE 17 SEQ ID Primer Sequence Location NO: UG_574_16110 CTGTCATGCCATCG5′PvuI 48 BtsI_rev TAAGATGCT Integration Site UG_575_16110GCGGCCAACTTACT 49 BtsI_for TCTGACAACG UG_576_16110 AAGGCGAGTTACA 3′PvuI50 AceIII_rev TGATCCCCCAT Integration Site UG 577 16110 CACCACGATGCCTG51 AceIII_for TAGCAATGG

TABLE 18 The iPCR for 5′iPCR conditions are described as follows.Initial Denaturation 94° C. 7 min Denaturation 94° C. 45 sec 28 cyclesAnnealing 60° C. 45 sec Elongation 68° C. 4 min Final Elongation 68° C.7 min Short storage  4° C.

Five microliter of PCR fragment was mixed with Orange G (5 μl) anddouble distilled water (10 μl) and loaded on agarose-gel to verify thedistribution of fragments. For 5′ iPCR of MseI and MluCI cuttedfragments good results were obtained and no unspecific band in controllanes was detected.

5′ amplicons of each sample (cut with MseI and MluCI) were combined andpurified with high pure PCR purification kit (Roche Diagnostics GmbH,Mannheim, Germany) in 40 μl Elution buffer.

Quantitative Real-Time Polymerase Chain Reaction

For each qPCR the non-specific fluorescent dye LightCycler®480 SYBRGreen I Master in the LightCycler®480 Instrument II System (RocheDiagnostics GmbH, Mannheim, Germany) was used. SYBR Green is excitedusing blue light (λ_(max)=488 nm) and it emits green light (λ_(max)=522nm). The SYBR Green dye binds to all double-stranded DNA in PCR. As aresult the fluorescence intensity and the amount of DNA product increasesimultaneously, which can be detected by the LightCycler® System aftereach cycle. For quantification the fluorescence was plotted against thenumber of cycles on a logarithmic scale. Slightly above the emittedbackground the threshold for detection of DNA-based fluorescence was setby the LightCycler® System. The number of cycles at which thefluorescence passes the threshold is termed quantification cycle (Cq)(Bustin, S. A., et al., Clin. Chem. 55 (2009) 611-22). During theexponential amplification phase a doubling of target DNA is expected inevery new cycle. However, the efficiency of amplification is oftenvariable among primers and templates. Analysis of the meltingtemperature of amplified DNA fragments gives first hints of thespecificity of used primer pairs and the amount of primer dimers.

An efficiency of primer template combination can be assessed by atitration experiment to create a standard curve or by efficiencycalculation program like LinRegPCR. The program uses non-baselinecorrected data from the LightCycler® System, performs a baselinecorrection on each sample separately, determines a window-of-linearityand then uses linear regression analysis to fit a straight line throughthe PCR data set. From the slope of this line the PCR efficiency of eachindividual sample is calculated.

For relative quantification the amount of target sequence were comparedto a reference sequence, by subtracting Cq (target) from Cq (reference).This normalization is termed ΔCq-method (Schefe, J. H., et al., J. Mol.Med. (Berlin) 84 (2006) 901-910). The reference sequence has to havevery stable values, considerably higher than the background values(Mock). Therefore for each reference sequence, the average and thecoefficient of variation of all samples within one project wascalculated and compared to background levels. Cq values of the differentconditions were displayed as % of the input sample, visualizing thedistance to mock control.

% of Input Sample=100*2^(ΔCq(Input Sample-ChIP Sample))

Regarding the large number of samples, all qPCR were accomplished in 384well plates. In each well following ingredients were transferred.

TABLE 19 Volume (μl) 384 well plate Solution 5 Sybr Green Master Mix 0.5Primer (10 pM) for. 0.5 Primer (10 pM) rev. 1.5 H2O 2.5 DNA (X ng/μl) 10Total

TABLE 20 The qPCR program was designed as follows: Temperature Grad.Appliance (° C.) Time (° C./sec) Cycles Pre- 95 10 min 4.8 1Denaturation Amplification 95 10 sec 4.8 40 60 5 sec (single) 2.5 72 15sec 4.8 Melt curve 95 5 sec 4.8 1 70 1 min 2.5 95 (continuously) cooling

In the case of the relative quantification of histone modifications, thenormalization of sample (treated) to input sample (untreated) isdirectly replaced by the normalization of treated target sequence totreated reference sequence. Further calculation of histone modificationper histone makes the normalization to input sample redundant.

The relative amount of specific histone modification close to human CMVmajor immediate-early promoter/enhancer fragment were estimated with theLivak method, also known as delta delta Cq (ΔΔCq) method (Livak, K. J.and Schmittgen, T. D., Methods 25 (2001) 402-408), as long asprimer-template efficiencies are close to 2 and close to each other (<2%difference of Cq mean).

The method can be used for relative quantification of a target regardingto a reference. The relative quantification of histone 3 acetylation andhistone 3 levels close to human CMV major immediate-earlypromoter/enhancer fragment were normalized to the histone 3 acetylationand the histone 3 levels close to reference gene in two steps. First ΔCqwas calculated as follows:

ΔCq=control−sample

ΔCq is the distance of quantification cycles (Cq) between control (e.g.ChIP sample value of Gusb) and target (e.g. ChIP sample value ofhCMV-MIE) of the same sample, amplified with different primer pairs. Toidentify the level of histone 3 modification per Histone 3, the ΔΔCqmethod was used.

$\left. {{\Delta\Delta}{Cq}}\rightarrow{ratio} \right. = \frac{2^{{\Delta{Cq}}_{target}({{control} - {sample}})}}{2^{{\Delta{Cq}}_{ref}({{control} - {sample}})}}$

Sequencing Methods

Sanger sequencing was performed by the company SequiServe (SequiServeGmbH, Vaterstetten, Germany).

Next generation sequencing was performed by GATC (GATC-biotech,Konstanz, Germany) and Active Motif (La Hulpe, Belgium) using IlluminaSequencing technology.

Protein Quantification

Protein concentrations were estimated in comparison to a proteinstandard with the Pierce® BCA protein assay kit (Thermo Scientific,Rockford, USA).

Protein Fractionation in Compartments

Proteins were fractionated in membrane/cytoplasmic and nuclear proteins(Misawa, Y, 2006). Following this CHO cells (1×10⁷ cells per sample)were sedimented by centrifugation (300×g, 3 minutes at 4° C.) and thepellet was washed twice in ice-cold PBS supplemented with Roche Complete(Roche Diagnostics GmbH, Mannheim, Germany). Washed pellet was lysedwith 1 ml 0.5% Triton X-100 lysis buffer and incubated on ice for 15minutes. Insoluble nuclei were separated by centrifugation (13,000 rpm,15 minutes at 4° C.). Membrane/cytoplasm containing supernatant wastransferred into a new tube and chilled on ice. The nuclear pellet wasrinsed with lysis buffer once and resuspended in 300 μl lysis buffercontaining 0.5% SDS followed by sonication (5 seconds, Output 2, dutycycle 90%, Branson Sonifier 450, Dietzenbach, Germany). Seven hundredmicroliter lysis buffer were added and sample was centrifuged at 13,000rpm, 15 minutes at 4° C. Nuclei containing supernatant were transferredinto new tube and protein fractions were stored at −80° C.

SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)

For denaturing SDS polyacrylamide gel electrophoresis NuPAGE gels(4-12%) were used (NuPAGE® Novex® 10% Bis-Tris Gel 1.0 mm×12 well, Cat.No. NP0302BOX, Invitrogen, Germany) in combination with the NuPAGE®electrophoresis system. According to Quick Reference Card (NuPAGE®Bis-Tris Mini Gels), gel was fixed in chamber and the inner cassette wasfilled with 1×MOPS running buffer (20× NuPAGE MOPS SDS Running Buffer,Cat. No. NP0001, Invitrogen, Germany). The outer chamber was also filledto two-thirds of its volume. The comb was removed and the lanes werepurged with a small pipette. 30 μl of sample was added to 10 μl 4×NuPAGE LDS-sample buffer, (Cat. No. NP0007, Invitrogen, Germany), 2 μl 1M DTT (final concentration 50 mM) and incubated at 95° C. for 5 minutes.Samples (20 μl/lane) and markers ((10 μl/lane) SeeBlue Markerprestained, Cat. No. LC 5625, Invitrogen; (5 μl/lane) MagicMark XP antiIgG, Cat. No. LC5602, Invitrogen, Germany) were transferred into thelanes and gel was applied at 30 volts for 50 minutes with NuPAGE®electrophoresis system.

SDS-PAGE and Western Blot

SDS-PAGE induced separated proteins were blotted to a methanol-activatednitrocellulose membrane according to NuPAGE protocol for denaturingelectrophoresis (NuPAGE® Technical Guide, Manual part no. IM1001,Invitrogen, Germany) in an XCell II Blot-Module (Invitrogen, Germany)with the appropriate reagents. Blotting was conducted in the SDS-PAGEchamber at 30 volt for 50 minutes. Blotted protein membrane wastransferred into a new chamber and washed 3 times for 10 minutes in1×TBS wash buffer (10×TBS: 0.5 M Tris-Base, 1.5 M NaCl, pH 7.5) followedby 5 minutes incubation in Ponceau solution (0.1%) for general proteindetection. The Ponceau solution was discarded and residues were washedaway with a 1 minute TBS wash steps twice.

Membrane was blocked for 1 hour at 20-30° C. in 1% blocking solution(Blocking solution: 1/10 Casein (Roche Diagnostics GmbH, Mannheim,Germany), 1×TBS). Subsequently membrane was incubated in primaryantibody solution (1% blocking solution+antibody) at 4° C. overnightwhile shaking. The membrane was washed three times in 1×TBST for 10minutes at 20-30° C. followed by a 1 minute wash step in 1×TBS. Themembrane was incubated in secondary antibody solution (1% blockingsolution+antibody) for 45 minutes at 20-30° C. or at 4° C. overnightwhile being agitated. The membrane was washed three times in 1×TBST for10 minutes at 20-30° C. followed by two times 1 minute wash step in1×TBS to remove Tween.

TABLE 21 Dilution Antibody name (WB) Host Company Cat. No. H4K16ac-ab1:5000 Rabbit Millipore 07-329 Anti GFP 1:2000 Mouse SIGMA G 6539 AntiFlag  1:1000- Mouse SIGMA F 3165 10000 Gal4(DBD + O1679 1:100-  RabbitSanta Cruz Sc-577 1000 Biotech Alpha-tubulin  1:10000 Mouse SIGMA T 9026Goat anti rabbit 1:2000 Goat Millipore 12-348 Goat anti mouse 1:2500Goat Millipore

Detection of Antibody-binding was done with the Lumi-LightPLUS westernblotting system (Roche, Penzberg, Germany).

Thawing of CHO Cell Lines

Falcon tubes were prepared with 5 ml of an appropriate medium. Cryovialswere stored on dry ice until thawing in a water bath (37° C.).Afterwards CHO cells were taken up into prepared falcon tubes andcentrifuged for 3 minutes at 500×g to remove residual DMSO. Pellet wasresuspended in 5 ml medium and propagated in disposable 125 ml shakeflasks (contain 20 ml appropriate medium) under standard humidifiedconditions (95% rH, 37° C., and 5% to 8% CO₂) at a constant agitationrate of 120 rpm/min to 150 rpm/min. Viability (>95%) and cellconcentration can be tested with Cedex HiRes Analyzer (Roche DiagnosticsGmbH, Mannheim, Germany).

Cultivation of CHO Cell Lines

Non-transfected CHO cells were split every 3-4 days and seeded with aconcentration of 2-3×10⁵ cells/ml in cultivation medium. Transfectedcells were seeded with various concentrations of methotrexate (MTX) ormethionine sulfoximine (MSX) as the selection agent. After recovery ofcell viability (3-4 weeks), stably transfected cells were selected inthymidine and protein-free medium containing 20 nM to 1200 nMMethotrexate (MTX) or 140-160 pM methionine sulfoximine (MSX) as theselection agent. The cells were propagated in 125 ml vented shake flasksunder standard humidified conditions (95% rH, 37° C., and 5% C02) at aconstant agitation rate of 120 rpm/min to 150 rpm/min. Every 3-4 daysthe cells were split into fresh mediums with a cell concentration of2-3×10⁵ cells/ml. Density and viability of the cultures were determinedusing the CASY TT or Cedex HiRes cell counter (Roche Innovates AG,Bielefeld, Germany).

Transient and Stable Transfection

Transfection was done with the Amaxa® Cell line Nucleofector® Kit V(Lonza, Cologne, Germany) and the transfection platforms Nucleofector™2bDevice and 96-well Shuttle™ System (Lonza, Cologne, Germany).

5×10⁶ CHO cells per transfection were centrifuged (200×g, 5 minutes) andthe supernatant was discarded. The pellet was resuspended in 100 μl ofsupplemented Nucleofector Solution V and 1.2 pmol of sterile plasmid wasadded by pipetting up and down. Plasmid was linearized for stabletransfection, for transient transfection the circular form wasmaintained. Suspension was mounted into bubble-free cuvette and programU-24 was activated for CHO cell line transfection. After pulse 500 μl ofpre-warmed media was mounted into cuvette and whole suspension wastransferred in 8 ml pre-warmed media. Cells were transferred intoincubator and cells were ready for first examinations two days after.

Cell Count with Cellavista

60 μl of CHO cell suspension was mixed with 60 μl trypan blue (0.1 μm)in 96 round bottom well and incubated at 20-30° C. for 5 minutes.Afterwards treated CHO cell suspension was 1:50 diluted with medium and200 μl were transferred into 96 flat bottom wells (Greiner, Germany).Slow centrifugation (500×g, 5 minutes) resulted in fast sedimentation ofcells.

For a precise cell count pictures from the middle of the well weretaken, to avoid display error of pictures approaching the edge of eachwell. The Cellavista cell imager (SynenTec, Munich, Germany) was usedfor the purpose of cell count. Each sample was done in replicate and theentire 10 pictures were used to calculate cell count.

Cell Count with Cedex HiRes Analyzer

For cell count of a sample number up to 40, the Cedex HiRes Analyzer(Roche, Germany) was used. Following this, the CHO cells were diluted ata ratio of 1:5 in HiRes medium and 300 μl were transferred to tubes.Cell calculation was done according to manufacturer's recommendations.Samples were analyzed in duplicates to verify the cell number.

Sample Preparation for Antibody Analysis

Cell concentration was calculated and 2 ml per sample were centrifuged(500×g, 5 minutes at 20-30° C.). Supernatant was transferred to new 96deep well plates and stored at −20° C. until use. Frozen supernatant wasthawed overnight at 4° C., 6× inverted and centrifuged (4,000 rpm, 30minutes at 20-30° C.). 310 μl were filtered with a multiscreen Milliporeplate atop a barcoded 96 round well plate by centrifugation (1,200 rpm,3 minutes at 20-30° C.).

Quantification of Antibody Production with HPLC

A chromatographic method was used to quantify the amount of antibodypresent in a sample. A Poros A column was used that binds the Fc-regionof the antibody. The antibody binds to the column and is subsequentlyeluted by low pH conditions. Protein concentration was established bydetermining the optical density (OD) at 280 nm, with a referencewavelength of 320 nm, using the molar extinction coefficient calculatedon the basis of the amino acid sequence.

Quantification of Antibody Production with ELISA

The ELISA (Enzyme Linked Immunosorbent Assay) technique is based on theantibody sandwich principle. A capture antibody specific to the analyteof interest for instance the Fc part of IgG is bound to a microtiterplate to create the solid phase. Following the blocking and washingsteps, samples, standards (dilution series of reference antibody), andcontrols are then incubated with the solid phase antibody, whichcaptures the analyte. After washing away unbound analyte, a conjugateddetection antibody (e.g. POD conjugated) is added. This detectionantibody binds to a different epitope of the molecule being measured,completing the sandwich. The BM Chemiluminescence ELISA Substrate POD(Roche Diagnostics GmbH, Mannheim, Germany) provides a substratesolution of peroxidase-based (POD, HRP) secondary detection system. Therate of signal generation in an immunoassay is directly proportional tothe amount of marker enzyme bound to the solid phase. Antibodyconcentration was calculated by the slope of standard dilution.

Generation of Recombinant CHO Cell Lines Comprising hCMV-MIE Promoter

Recombinant cell lines expressing human antibody constructs of class IgGwere generated by transient or stable transfection of CHO-K1 suspensiongrowing cells. Light and heavy chain expression cassettes of humanimmunoglobulin were both under the control of a human CMV majorimmediate-early promoter and enhancer.

The vector also comprised a nucleic acid sequence encoding murinedihydrofolate reductase (DHFR). Transfection of cells was performed byAmaxa nucleofection system (Lonza Cologne GmbH, Cologne, Germany).

CHO-K1 M suspension was transfected with circular plasmid DNA fortransient expression of antibody, using the Nucleofector device incombination with the Nucleofector Kit V (Lonza Cologne GmbH, Cologne,Germany) according to the manufacturer's protocols. Transienttransfected cell suspensions were seeded in 96 well plates and incubatedfor 5 days.

Stable transfected cell suspensions were seeded in 384 or 6-well platescontaining thymidine-free medium with 250 to 1600 nM methotrexate (MTX)as the selection agent. After three to four weeks antibody-expressingcell pools were examined for long term stability over a period of one tothree months. Antibodies expressing single cell clones were seeded in384 and 96 well plates. After three weeks, antibody-expressing celllines were identified by measuring antibody titers in the culture mediumby ELISA. Growing wells were randomly picked and in the interests oflong term stability assay cell clones were expanded in higher volumes (3ml per well in 6 well plates) and antibody concentration was determinedby protein A HPLC and ELISA at the end of each passage.

The cells were propagated in disposable 125 ml vented shake flasks or 6well plates under standard humidified conditions (95% rH, 37° C., and 5%to 8% CO₂) at a constant agitation rate of 120 rpm/min to 150 rpm/min.Every 3-4 days the cells were split into fresh mediums. Density andviability of the cultures were determined using the Cedex HiRes cellcounter (Roche Innovates AG, Bielefeld, Germany) or Cellavista CV3.1(SynenTec Bio Services GmbH, Munich, Germany).

Long-Term Cultivation and Production of CHO Cell Lines ComprisinghCMV-MIE Promoter

The cells were tested for phenotypic (i.e. production) stability for 2to 3 months after transfection in the presence of selection agent MTX.The cells were continuously cultivated in vented 125 ml shake flaskscontaining 20-40 ml medium or 6 well plates containing 2-4 ml mediumwith selection agent and diluted twice a week with fresh medium. Seedingdensity was 2 to 3*10⁵ cells/ml. Prior to passage, viable cell densityand viability were determined.

Antibody concentration of the supernatant (antibody titer) wasdetermined by protein A HPLC and ELISA at the end of each passage. Fromthese data, the cell specific productivity (qP) for each passage wascalculated using the following formula:

${qP} = \frac{{P2} - {P1}}{\left( {{D2} - {D1}} \right)/2*\Delta t}$

qP [pg/cell/d]: cell specific productivity,

P₁ [μg/ml]: antibody titer at the beginning of the passage,

P₂ [μg/ml]: antibody titer at the end of the passage,

D₁ [cells/ml]: viable cell density at the beginning of the passage,

D₂ [cells/ml]: viable cell density at the end of the passage,

Δt [d]: duration of the passage.

The qP values were plotted against the age of culture at the end of therespective passage in generations. A linear trend line was calculatedover all qP data points and the relative alteration of the qP (inpercent) over the period was calculated in house, according to thefollowing equation:

${\Delta qP} = \frac{m*a}{{qP}_{0}*100}$

ΔqP [%]: percentage alteration of qP,

m [pg/cell/d/generation]: slope of linear trend line,

a [no. of generations]: age of culture,

qP₀: y-axis intercept of linear trend line.

In regard to lower number of data points obtained for each sample theaverage of the last three qP values was divided by the average of thefirst two qP values and displayed in percent to obtain ΔqP.

${\Delta qP} = {\frac{{average}{qP}{EOS}}{{average}{qP}{PSB}}*100}$

Average qP EOS: average of last three qP values

Average qP PSB: average of the first two qP values

Treatment of Recombinant CHO Cell Lines, Used for Identification ofEpigenetic Marker

32 CHO cell lines of two projects were used to identify epigeneticmarkers, predicting target gene expression two months in advance. Forboth projects data including antibody concentration, C-425 methylationstatus of hCMV-MIE promoter and copy number were collected over a periodof 60 generations (˜2 months). Project H comprises 20 CHO-K1 cell linesexpressing a human antibody of class IgG under control of hCMV-MIEpromoter. The vector further comprises a nucleic acid sequence encodingglutamine synthetase, making cells susceptible to methionine sulfoximine(MSX) selection. Project T comprises 12 CHO-K1 cell lines expressing ahuman antibody of class IgG under control of hCMV-MIE promoter. Thosecell lines are prone to methotrexate (MTX) selection because of thetransgene of the murine dihydrofolate reductase. Frozen start cultureswere thawed and cultivated in appropriate mediums comprising 250 nM MTXor 140 pM MSX. After two weeks (time point PSB) of cultivation cellswere harvested and chromatin immunoprecipitation with followingantibodies was carried out.

TABLE 22 list for project T ChIP Target Name company grade host cat. no.H3K4me3 ChIPAb+ Trimethyl- Millipore v rabbit 17-614 Histone H3 (Lys4)monoclonal H3K27me3 ChIPAb+ Trimethyl- Millipore v rabbit 17-622 HistoneH3 (Lys27) polyclonal H3K9me3 Anti-Histone H3 Abcam v rabbit ab8898(trimethyl K9) antibody polyclonal H3 Anti-Histone H3 Abcam v rabbitab1791 antibody polyclonal H3ac Anti-acetyl-Histone H3 Millipore vrabbit 06-599 polyclonal

TABLE 23 list for H ChIP target Name company grade host cat. no. H3K4me3Histone H3K4me3 activemotif ✓ rabbit 39915 antibody (pAb) PolyclonalH3ac Anti-acetyl-Histone H3 Millipore ✓ rabbit 06-599 Antibodypolyclonal H3K9me3 ChIPAb+ Trimethyl- Millipore v polyclonal 17-625Histone H3 (Lys9) H3K27me3 ChIPAb+ Trimethyl- Millipore v rabbit 17-622Histone H3 (Lys27) Polyclonal H3 Anti-Histone H3 Abcam ✓ Rabbit ab1791antibody polyclonal

TABLE 24 Quantitative PCR was performed with the following primers. SEQID Primer NO: NO: Target Sequence 32 396 hCMV-MIE forward TACATCAATGGGCG1 TGGATA 33 397 hCMV-MIE reverse AAGTCCCGTTGATT 1 TTGGTG 34 386Gusb forward 1 CAGGGTGGGATGCT CTTC 35 387 Gusb reverse 1 GCCGGTTTTCCGAGAAGT 36 431 Eifi forward 1 GTTCCCGGCACTGA CACT 37 432 Eif3i reverse 2ACTTGATCTGCGTGAT GGAC 38 484 Fox2a forward 1 ATCACCCGTACTGCT GCTCT 39485 Fox2a reverse GAGGCTTCTGGGGAT CTCTT 40 468 Gata5 forward 1CACCTACCCCATCCT GTCTG 41 469 Gata5 reverse 1 GAGGAGGTGAAGGC AAAGTCT 42388 Rho forward 1 AGCCTCGGTCTCTA TTGACG 43 389 Rho reverse 1CGTTGGAGAAGGGC ACATAA 52 474 UNCI3c forward 1 GGGTGCTTTACGGA AACTGA 53475 UNCI3c reverse 1 GCTTCTTATGCCCCA GGTTT

Transgene Copy Number Determination

Transgene copy number examination was performed as previously described(Osterlehner, et al. 2011). Transgene copy per sample (LCs or HCs) wasextrapolated from a standard curve of a dilution series. Assuming thateach cell contain approximately 6 μg DNA the number of transgenes percell (LC_(cell)) was calculated as follows.

$\begin{matrix}{{LC}_{cell} = {\frac{{LC}_{s}}{50000}*6}} & {{HC}_{cell} = \frac{{HC}_{s}}{50000}}\end{matrix}*6$

Computational Analysis with JMP

Percentage methylation of C-179 and acetylation per histone 3 close tohCMV-MIE at begin of cultivation phase were plotted against thealteration over 60 generations of specific productivity (ΔqP) in astandard least squares fit model. Therefore software JMP10 (JMP® 10.0.1Release: 2, 64-bit edition; SAS Institute Inc.) was used and P-values ofeffect leverage plot were calculated. In addition outlier analyses wereperformed by employing the Jackknife technique. The LogWorth statisticwas performed to identify the best split node to subdivide cellpopulations by stability and degree of epigenetic modification (Sall2002).

EXAMPLE 2

Generation of Recombinant CHO Cell Lines

Recombinant cell lines expressing a human antibody of class IgG weregenerated by stable transfection of CHO-K1 or CHO-DG44 suspensiongrowing cells with a vector encoding the light and heavy chain ofantibody comprising a human immunoglobulin kappa light chain and a humanimmunoglobulin gamma 1 or gamma 4 heavy chain. The vector furthercomprised a nucleic acid encoding murine dihydrofolate reductase (DHFR)or glutamine synthetase (GS). Light and heavy chain expression cassetteswere both under the control of a human CMV major-immediate-earlypromoter and enhancer (SEQ ID NO: 01). Transfection of cells was eitherperformed by nucleofection (Lonza Cologne GmbH or Amaxa Biosystems) orby electroporation using Gene Pulser XCell (BIO-RAD).

For example, CHO-K1 or CHO-DG44 suspension were transfected withlinearized plasmid DNA, using the Nucleofector device in combinationwith the Nucleofector Kit V (Lonza Cologne GmbH, Cologne, Germany) or byelectroporation using the Gene Pulser XCell (Bio-Rad, Hercules, Calif.),according to the manufacturers' protocols. Transfected cells were seededinto 96 or 384-well plates containing thymidine-free medium with variousconcentrations of methotrexate (MTX) as selection agent or methioninesulfoximine (MSX) as selection agent. After three weeks,antibody-expressing cell lines were identified by measuring antibodytiters in the culture medium by ELISA. Top producers were expanded tohigher volumes, subcloned by limiting dilution and cryoconserved.

Stably transfected cells were selected in thymidine and protein freemedium containing 20 nM to 1200 nM Methotrexate (MTX) or 140-160 pMmethionine sulfoximine (MSX) as selection agent. Antibody expressingcells or cell lines were identified by measuring antibody titers in theculture medium and subcloned by limiting dilution and/or FACS singlecell deposition.

The cells were propagated in disposable 50 ml or 125 ml vented shakeflasks under standard humidified conditions (95% rH, 37° C., and 5% to8% CO₂) at a constant agitation rate of 120 rpm/min to 150 rpm/min.Every 3-4 days the cells were split into fresh medium. Density andviability of the cultures were determined using the CASY TT or CedexHiRes cell counter (Roche Innovates AG, Bielefeld, Germany).

Furthermore, standard cell culture techniques were applied as describede.g. in Current Protocols in Cell Biology, Bonifacino, J. S. et al.(Eds), John Wiley & Sons, Inc., New York (2000).

EXAMPLE 3

Long-Term Cultivation and Production

CHO cell lines obtained according to Example 2 were investigated forlong-term productivity.

The cells were tested for phenotypic, i.e. production, stability for 35to 70 generations in the absence of a selection agent. The cells werecontinuously cultivated in vented 125 ml shake flasks containing 50 mlmedium without selection agent and diluted twice a week with freshmedium. Seeding density was 2 to 3×10⁵ cells/ml. Prior to passage viablecell density and viability were determined. The age of the culture ingenerations at the end of each passage was calculated according to thefollowing equitation:

a ₂ =a ₁+ln(D ₂ /D ₁)/ln 2  (Formula 1)

with

-   -   a₂ [no. of generations]: age of the culture at the end of the        passage,    -   a₁ [no. of generations]: age of the culture at the beginning of        the passage i.e. age of the culture at the end of the previous        passage,    -   D₁ [cells/ml]: viable cell density at the beginning of the        passage,    -   D₂ [cells/ml]: viable cell density at the end of the passage.

Antibody concentration in the supernatant (antibody titer) wasdetermined by protein A HPLC at the end of each passage. From thesedata, the specific production rate (SPR) for each passage was calculatedusing the following formula:

SPR=P ₂ −P ₁/((D ₂ +D ₁)/2*Δt)  (Formula 2)

with

-   -   SPR [pg/cell/d]: specific production rate,    -   P₁ [μg/ml]: antibody titer at the beginning of the passage,    -   P₂ [μg/ml]: antibody titer at the end of the passage,    -   D₁ [cells/ml]: viable cell density at the beginning of the        passage,    -   D₂ [cells/ml]: viable cell density at the end of the passage,    -   Δt [d]: duration of the passage.

The SPR values were plotted against the age of culture at the end of therespective passage in generations. A linear trend line was calculatedover all SPR data points and the relative alteration of the SPR (inpercent) over the period tested was calculated according to thefollowing equitation:

ΔSPR=m*a/SPR₀*100  (Formula 3)

with

-   -   ΔSPR [%]: percentual alteration of SPR,    -   m [pg/cell/d/generation]: slope of linear trend line,    -   a [no. of generations]: age of culture,    -   SPR₀: y-axis intercept of linear trend line.

Almost all the cell lines showed a decrease in productivity, whereby theloss of productivity tends to be stronger under condition withoutselection agent

TABLE 25 Change in SPR during cultivation of five cell lines in thepresence and/or absence of selection agent. Selection Agent MTX parentalcell line ΔSPR ΔSPR CHO-K1 (−) MTX (−) MTX Sample No. 30 generations 60generations K18.1 −59% n.d. G25-10 −29% −57% G25-17 −73% n.d. G42-5  0% −1% Selection Agent MTX parental cell line ΔSPR ΔSPR CHO-DG44 (−) MTX(−) MTX Sample No. 30 generations 60 generations 43-16 A10 −49% n.d.Selection Agent MSX parental cell line ΔSPR ΔSPR CHO-K1 (+) MSX (−) MSXSample No. 60 generations 60 generations H-1 −47% −80% H-2 −71% −70% H-3−73% −90% H-4 −69% −70% H-5 −40% −49% H-6 −80% −72% H-7 −66% −82% H-8−67% −76% H-9 −87% −77% H-10 −16% −73% H-11 −43% −96% H-12 −42% −33%H-13  8% −19% H-14  7%  5% H-15 −17% −40% H-16  16% −33% H-17 −13% −31%H-18 −68% e.i.m. H-19 −11%  3% H-20 −40% −81% Selection Agent MTX HostCell line ΔSPR ΔSPR CHO-K1 (+) MTX (−) MTX Sample No. 60 generations 60generations T-1 −35% −34% T-2 −31% −72% T-3 −82% −97% T-4 −23% −47% T-5−16% −22% T-6 −14% −20% T-7 −16% −32% T-8 −65% −79% T-9 −28% −49% T-10 4%  1% T-11 −10% −22% T-12 −32% −23% n.d. = not determined. e.i.m. =error in measurement

EXAMPLE 4

Identification of Methylated CpG Sites within Human CMVMajor-Immediate-Early Promoter/Enhancer DNA by Bisulfite Treatment andDNA Sequencing

The human CMV major-immediate-early promoter/enhancer fragment (SEQ IDNO: 01) used for the expression of antibody light and heavy chain genescontains 33 CpG sites.

Genomic DNA was isolated from the CHO cell lines K18.1, G25-10, G25-17,G42-5 and 43-16 A10 using the Allprep DNA/RNA Mini Kit from Qiagen(Hilden, Germany). Five microgram DNA was cleaved with the enzyme DraIand quantified by measuring the extinction at 260 nm. One hundrednanogram DNA was subjected to bisulfite treatment and purified using theEpiTect Bisulfite Kit (Qiagen, Hilden, Germany). Bisulfite treated DNAwas recovered in 20 μl RNAse-free water (Qiagen, Hilden, Germany).

In order to amplify strand A (forward) of the human CMVmajor-immediate-early promoter/enhancer fragment (SEQ ID NO: 02), 1 μlbisulfite treated DNA was combined with 24 μl PCR master mix andsubjected to PCR using the GeneAmp® PCR System 9700 (Applied BiosystemsInc., USA).

24 μl PCR master mix comprised:

-   -   1 μl forward primer 227 of SEQ ID NO: 06 (10 pmol/μl),    -   1 μl reverse primer 229 of SEQ ID NO: 08 (10 pmol/μl),    -   22 μl Platinum© PCR SuperMix HighFidelty (Invitrogen Corp.,        USA).

Forward primer 227 is complementary to the 5′-end of the human CMVmajor-immediate-early promoter/enhancer fragment. Reverse primer 229binds downstream within the 5′-UTR of the immunoglobulin genes.

The PCR conditions were as follows:

TABLE 26 temp. duration Step 1 Denaturation 95° C. 10 min. Step 2: PCRDenaturation 94° C. 30 sec. # cycles: 45 Annealing 50° C. 2 min.Extension 68° C. 2 min. Step 3 Final Extension 72° C. 10 min. Step 4Soak  4° C. indefinite

The PCR product was checked for size and purity by agarose gelelectrophoresis and cloned in the vector pCR4 (Invitrogen Corp., USA)using the TOPO TA cloning kit (Invitrogen Corp., USA). Plasmid cloneswere isolated and analyzed by restriction digest and agarose gelelectrophoresis. For each cell line, 19 to 22 plasmids containing theinsert were sequenced. In order to estimate the deamination efficiencyof the bisulfite treatment at non CpG sites, the number of residualcytosine at non-CpG sites was determined. The percentual deaminationefficiency was calculates as follows:

E _(mod)=100−(C _(res) /C _(total)*100)  (Formula 4)

with

-   -   E_(mod)[%]: deamination efficiency,    -   C_(res): number of residual cytosine at non-CpG sites in all        inserts analyzed, PCR primers sites excluded,    -   C_(total): number of cytosine in the non-bisulfite treated CMV        promoter/enhancer fragment, PCR primer sites excluded,        multiplied by the number of inserts analyzed, i.e. 107*20.

It was found that the deamination efficiency at non-CpG cytosine wasgreater than 99% in all samples (Table 12).

TABLE 27 Deamination efficiency at non-CpG cytosine. Cell Cell Cell CellCell line line line line line 43-16 K18.1 G25-10 G25-17 G42-5 A10E_(mod) 99.1% 99.3% 99.5% 99.3% 99.3%

Protection of 5-methyl cytosine from deamination was confirmed bybisulfite treatment and subsequent cloning and sequencing of plasmid DNAisolated from dcm⁺ E. coli. Dcm⁺ E. coli methylate the internal cytosineresidues within the sequences CCAGG or CCTGG (dcm-sites). It was foundthat the deamination efficiency was 99% at non-dcm-sites and less than5% at internal cytosine within dcm-sites.

To quantify the extend of DNA methylation at CpG sites within thebisulfite treated CMV promoter/enhancer fragment, the number of cytosinefound at each CpG-site was determined and plotted for each analyzedcell.

Cell line K18.1 is highly methylated (FIG. 3A). The frequency ofmethylation accumulates in 3 clusters, one at the 5′-end, one the 3′-endand one at around position 400. The highest degree of methylation wasfound at position 425. Fourteen out of the twenty-two inserts sequencedhad a cytosine here.

Methylation of the CMV promoter from cell line 43-16 A10 was noticeable(FIG. 3E). The distribution of methylation was similar to thedistribution observed with cell line K18.1. Position 425 was methylatedmost often. Five of the twenty inserts sequenced contained a cytosine inthis position.

In the three other cell lines investigated cytosine were detected onlysporadically at CpG sites (FIGS. 3B, 3C and 3D).

EXAMPLE 5

Quantitative Methylation Specific PCR of Bisulfite Treated Human CMVMajor-Immediate-Early Promoter/Enhancer DNA

In this example a methylation specific real-time qPCR as method todetect methylation at a CpG position, to be more precise at position 425of the hCMV promoter nucleic acid, is reported.

Two sets of primers were designed:

-   -   a methylation specific primer pair (MSP primer pair) selectively        amplifying deaminated CMV promoter DNA with a cytosine in        position 425 representing DNA that is methylated at position        425, and    -   a universal primer pair amplifying deaminated CMV promoter DNA        irrespective of the methylation status.

The universal primer pair was used for normalization. To be used in thesame PCR run both primer pairs should have similar melting points.

The designing of primers sensing methylation at position 425 wascomplicated by the presence of two additional CpG sites in closeproximity (position 416 and position 437). Methylation sensitive primersshould detect 5mC425 independent of the methylation status of position416 and position 437.

Four deaminated human CMV major-immediate-early promoter/enhancerfragments isolated in Example 4 representing the possible sequencevariations in positions 425 and 437: #11, #62, #01 and #04 (Table 28,SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22) have beenused as qPCR templates for methylation specific PCR and universal primerpairs.

TABLE 28 Expected results for MSP and universal primer pairs in qPCR.Templates #11 #62 #01 #04 Pos. 425 T C C T Pos. 437 T C T CAmplification MSP primer pair — + + — Universal primer pair + + + +

With the universal primer all four templates can be comparably amplifiedwhereas the methylation specific primer pair can selectively amplifytemplate #62 and template #01. ΔCp should be as small as possiblebetween the methylation specific primer pair and the universal primerpair on templates #62 and template #01. Cp values obtained with themethylation specific primer pair on template #11 and template #04 shouldbe as high as possible, i.e. ΔCp compared to amplification with theuniversal primer pair should be maximal.

For qPCR the LightCycler® 480 II system was employed (Roche DiagnosticsGmbH, Mannheim, Germany) and samples were prepared using theLightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim,Germany). Five microliters template solution containing 0.05 ng DNA wascombined with 15 μl PCR master mix in a well of a 96-well multi wellplate.

15 μl PCR master mix comprised:

-   -   4.2 μl water,    -   0.4 μl forward primer 239, 263 or 264 (also possible primer 227,        228, 240, 238) (10 pmol/μl),    -   0.4 μl reverse primer 237, 254, 262, 265, 266, 267 or 268 (also        possible primer 229) (10 pmol/μl),    -   10 μl SYBR Green I Master.

The multi well plate was sealed with a LightCycler® 480 sealing foil(Roche Diagnostics GmbH, Mannheim, Germany) and centrifuged at 1,500×gfor 2 minutes. Afterwards the plate was mounted into the LightCycler®480 system and subjected to qPCR. Each sample was tested in duplicate,triplicate or quadruplicate. To allow determination of absolute copynumbers, standard curves were generated for the LC and the HC transgene,using the linearized expression plasmid as standard. Standard dilutionscontained 2.5×10⁷, 2.5×10⁶, 2.5×10⁵, 2.5×10⁴ or 2.5×10³ plasmid copies.Genomic DNA was tested in triplicate; standards were run inquadruplicate.

TABLE 29 The used PCR conditions were: ramp No. of T t rate acqui- stepcycles [° C.] [min:s] [° C. s-1] sition de-  1 95 10:00 4.40 —naturation real-Time denaturation 45 95 00:10 4.40 — PCR annealing 57 to59 00:15 2.20 — elongation 72 00:07 4.40 — detection 66 00:01 2.20single melting denaturation  1 95 00:05 4.40 — curve annealing 60 01:002.20 — melting 90 00:00 0.11 contin- uous cooling  1 40 00:30 2.2 

The collection and analysis of the data was done with the LightCycler®480 software version 1.5. The apparent degree of methylation wascalculated using the following formula, where ideal amplificationefficiency was assumed (E=2).

mC _(app)=2^(Cp(t)-Cp(m)*)100  (Formula 5)

with

-   -   mC_(app) [%]: apparent degree of methylation,    -   Cp(t): Cp value obtained with universal primers,    -   Cp(m): Cp value obtained with methylation-specific primers.

During primer evaluation, it was found that designing methylationspecific primer, which are highly selective for deaminated CMV promoterDNA with a cytosine at position 425 (“methylated”), has to be performedwith care. Primer 266 and 267 showed the maximal difference betweenCp(#11) and Cp(#62), i.e. the highest selectivity for “methylated” DNA.Primer 265, 268 and 254 showed minor selectivity. The universal primerpair 263/237 was tested as control for minimal ΔCp (FIG. 6 and Table30).

TABLE 30 Results of primer evaluation. ΔCp Stdev. ΔCp Mean STD [Cp(#11)-[Cp(#11)- Primer Template Cp Cp Cp(#62)] Cp(#62)] 263 + 237 #11 11.710.06 0.23 0.06 #62 11.48 0.01 263 + 254 #11 15.22 0.08 3.89 0.19 #6211.32 0.17 263 + 265 #11 20.29 0.23 8.74 0.23 #62 11.55 0.02 263 + 266#11 23.76 0.04 12.31 0.16 #62 11.45 0.16 263 + 267 #11 24.74 0.14 13.050.16 #62 11.69 0.07 263 + 268 #11 21.75 0.06 10.25 0.10 #62 11.51 0.09

In FIG. 5 the results obtained with the methylation specific primer pair239/267 (SEQ ID NO: 11, SEQ ID NO: 18) in combination with the universalprimer pair 239/237 (SEQ ID NO: 11, SEQ ID NO: 09) is shown. Universalprimer pair 239/237 amplified all four templates about equally well,whereas the methylation specific primer pair 239/267 amplified templates#62 and #01. Templates #11 and #4 are only poorly amplified by theprimer pair 239/267.

Methylation frequency calculated from the Cp values was almost 100% fortemplate #62 and template #01 and almost 0% for template #11 andtemplate #04 (Table 15). This shows that methylation specific primerpair 239/267 in combination with the universal primer pair 239/237 canbe used to discriminate CMV promoter DNA which is methylated at position425 from CMV promoter DNA which is non-methylated at position 425 byreal-time qPCR.

Additional universal and methylation specific primer pairs were foundand are characterized in Table 31 and 32.

TABLE 31 Methylation specific reverse primer 267 and non-methylationspecific reverse primer 237 combined with three different forwardprimers. methylation universal primer pairs site 239 (for) + 237 (rev)263 (for) + 237 (rev) 264 (for) + 237 (rev) template 425 437 Cp(u) StdevCp(u) Stdev Cp(u) Stdev # 11 T T 16.41 0.23 15.65 0.01 15.73 0.1  # 62 CC 16.73 0.04 16    0.08 16.38 0.25 # 01 C T 17.51 0.24 16.64 0.08 16.870.21 # 04 T C 17.58 0.24 16.18 0.16 16.54 0.22 methylation C425 specificprimer pairs site 239 (for) + 267 (rev) 263 (for) + 267 (rev) 264(for) + 267 (rev) template 425 437 Cp(m) Stdev Cp(m) Stdev Cp(m) Stdev #11 T T 30.71 0.23 26.84 0.28 28.01 0.14 # 62 C C 16.74 0.13 16    0.3816.82 0.29 # 01 C T 17.52 0.18 16.91 0.36 17.06 0.17 # 04 T C 25.34 0.2423    0.14 23.68 0.27 methylation site ΔCp = Cp(m) − Cp(u) template 425437 ΔCp Stdev ΔCp Stdev ΔCp Stdev # 11 T T 14.3  0.33 11.19 0.28 12.280.17 # 62 C C  0.01 0.14  0    0.39  0.44 0.38 # 01 C T  0.01 0.30  0.270.37  0.19 0.27 # 04 T C  7.76 0.34  6.82 0.21  7.14 0.35 methylationsite mCapp [%] template 425 437 mCapp Stdev mCapp Stdev % rel. Stdev #11 T T  0.0  0.0   0.0  0.0  0.0  0.0 # 62 C C 99.3  9.4 100.0 26.9 73.719.6 # 01 C T 99.3 20.7  82.9 21.2 87.7 16.4 # 04 T C  0.5  0.1   0.9 0.1  0.7  0.2

TABLE 32 Methylation specific reverse primer 266 and non-methylationspecific reverse primer 237 combined with two different forward primers.methylation universal primer pairs site 263 (for) + 237 (rev) 264(for) + 237 (rev) template 425 437 Cp(u) Stdev Cp(u) Stdev # 11 T T15.32 0.18 15.88 0.08 # 62 C C 15.73 1.07 16.22 0.57 # 01 C T 15.42 0.1916.35 0.22 # 04 T C 15.4  0.24 16.8  1.12 methylation C425 specificprimer pairs site 263 (for) + 267 (rev) 264 (for) + 267 (rev) template425 437 Cp(m) Stdev Cp(m) Stdev # 11 T T 28.33 0.62 28.27 0.44 # 62 C C15.64 0.25 16.32 0.35 # 01 C T 15.83 0.34 16.59 0.01 # 04 T C 22.39 0.1522.83 0.88 methylation site ΔCp = Cp(m) − Cp(t) template 425 437 ΔCpStdev ΔCp Stdev # 11 T T   13.01 0.65 12.39 0.45 # 62 C C  −0.09 1.10 0.1  0.67 # 01 C T    0.41 0.39  0.24 0.22 # 04 T C    6.99 0.28  6.031.42 methylation site mCapp [%] template 425 437 mCapp Stdev % rel.Stdev # 11 T T   0.0  0.0  0.0  0.0 # 62 C C 106.4 81.1 93.3 43.3 # 01 CT  75.3 20.3 84.7 12.9 # 04 T C   0.8  0.2  1.5  1.5

For quantification of the degree of methylation over a broad rangetemplate #62 was mixed in different ratios with template #11. qPCR wasperformed as described above using primer pairs 239/237 and 239/267 andthe recovery of template #62 in the template #11 background wascalculated.

For calculation of the fraction of template #62 DNA, the amplificationefficiencies of the primer pairs under the used conditions weredetermined. Serial dilutions of templates #62 and #11 from n=0.005 ng to0.5 ng DNA were subjected to qPCR and the determined Cp values wereplotted against log (n). A linear regression line was calculated usingXLfit (Microsoft). The amplification efficiency was calculated using thefollowing formula:

E=10^(−1/m)  (Formula 6)

with

-   -   E: amplification efficiency,    -   m: slope of linear trend line.

The amplification efficiencies of both primer pairs were calculated tobe approximately 1.7. The following formula was employed for calculatingthe fraction of template #62 DNA:

mC=1.7^(Cp(t)-cp(m)*)100  (Formula 7)

with

-   -   mC [%]: fraction of DNA methylated at position 425,    -   Cp(t): Cp value obtained with universal primer pair,    -   Cp(m): Cp value obtained with methylation specific primer pair.

The determined fractions of template #62 DNA from two independentexperiments were plotted against the expected values (FIG. 7 ).Quantification of methylation between 1% and 100% can be performed.

EXAMPLE 6

Identification of Methylation Status of C-425 within Human CMVMajor-Immediate-Early Promoter/Enhancer DNA by Bisulfite Treatment andReal Time Quantitative PCR

Bisulfite Treatment of Genomic DNA of CHO Cell Lines

Assay was performed as described in Osterlehner et al. (supra). GenomicDNA was isolated from the CHO cell lines using the Allprep DNA/RNA MiniKit from Qiagen (Hilden, Germany). Five microgram DNA was cleaved withthe enzyme DraI and quantified by measuring the extinction at 260 nm.One hundred nanogram DNA was subjected to bisulfite treatment andpurified using the EpiTect Bisulfite Kit (Qiagen, Hilden, Germany).Bisulfite treated DNA was recovered in 20 μl RNAse-free water (Qiagen,Hilden, Germany).

In order to amplify strand A (forward) of the human CMVmajor-immediate-early promoter/enhancer fragment (SEQ ID NO: 02), 1 μlbisulfite treated DNA was combined with 24 μl PCR master mix andsubjected to PCR using the GeneAmp® PCR System 9700 (Applied BiosystemsInc., USA).

24 μl PCR master mix comprised

-   -   1 μl forward primer 227 of SEQ ID NO: 06 (10 pmol/μl),    -   1 μl reverse primer 229 of SEQ ID NO: 08 (10 pmol/μl),    -   22 μl Platinum® PCR SuperMix HighFidelty (Life Technologies,        Carlsbad, Calif.).

Forward primer 227 is complementary to the 5′-end of the human CMVmajor-immediate-early promoter/enhancer fragment. Reverse primer 229binds downstream within the 5′-UTR of the immunoglobulin genes.

TABLE 33 The PCR conditions were as follows: temp. duration Step 1Denaturation 95° C. 10 min. Step 2: Denaturation 94° C. 30 sec. PCR #Annealing 50° C. 2 min. cycles: 45 Extension 68° C. 2 min. Step 3 FinalExtension 72° C. 10 min. Step 4 Soak  4° C. indefinite

The PCR product was checked for size and purity by agarose gelelectrophoresis.

Methylation Specific Real Time Quantitative PCR

LightCycler 480 II system (Roche Diagnostics GmbH, Mannheim, Germany) incombination with LightCycler 480 SYBR Green I Master (Roche DiagnosticsGmbH, Mannheim, Germany) was used to perform qPCR. Fifty picogramplasmid DNA, 3 μl bisulfite-treated genomic DNA, or 5 μl of a 1:50,000dilution of the PCR product of primers 227 and 229 (SEQ ID NO: 06/08)with bislufite-treated genomic DNA were used as templates. For qPCR,templates were mixed with 4 or 12 pmol forward and reverse primer, 10 μlLightCycler 480 SYBR Green I Master, and nuclease-free water, to make upa total volume of 20 μl. To quantify the total human CMVmajor-immediate-early promoter/enhancer fragment DNA the primer pair239/237 (SEQ ID NO: 11/09) was used, the methylated DNA at positionC-425 (SEQ ID NO: 01) was detected with primer pair 239/267 (SEQ ID NO:11/18). The PCR mix was added into a multi well plate.

The multi well plate was sealed with a LightCycler® 480 sealing foil(Roche Diagnostics GmbH, Mannheim, Germany) and centrifuged at 1,500×gfor 2 minutes. Afterwards the plate was mounted into the LightCycler®480 system and subjected to qPCR. Each sample was tested in duplicate,triplicate or quadruplicate.

TABLE 34 PCR conditions were as follows: ramp No. of T t rate acqui-step cycles [° C.] [min:s] [° C. s-1] sition de-  1 95 10:00 4.40 —naturation real-Time denaturation 45 95 00:10 4.40 — PCR annealing 57 to59 00:15 2.20 — elongation 72 00:07 4.40 — detection 66 00:01 2.20single melting denaturation  1 95 00:05 4.40 — curve annealing 60 01:002.20 — melting 90 00:00 0.11 contin- uous cooling  1 40 00:30 2.2 

Data collection and analysis was performed with LightCycler 480 softwareversion 1.5 (Roche Diagnostics GmbH, Mannheim, Germany). The methylationof mC was expressed in percentage by following equation:

mC=E ^(Cq(t)-Cq(m))*100  (Formula 8)

Cq(t) being the quantification cycle obtained with the universal primerpair 239/237, Cq(m) represents the quantification cycle obtained withthe methylation-specific primer pair 239/267, and E being theamplification efficiency (MIQE Guidelines). E was approximately 1.7 for0.2 pmol/μl primer concentration and 2.0 for 0.6 pmol/μl primerconcentration.

EXAMPLE 7

Human CMV Major-Immediate-Early Promoter/Enhancer MethylationCorrelation with Long-Term Productivity

Methylation Specific qPCR with Pre-Amplified Human CMVMajor-Immediate-Early Promoter/Enhancer Strand a

As reported in Example 4 genomic DNA was isolated from CHO cell linesK18.1, G25-10, G25-17, G42-5 and 43-16 A10, cleaved with the enzyme DraIand deaminated by bisulfite treatment. The strand A of the human CMVmajor-immediate-early promoter/enhancer was amplified using primer 227and 229.

The PCR product was diluted 1:50,000. Five microliters of each dilutionwere used for real-time qPCR. Primer 239 and 237 were employed forquantification of total CMV promoter DNA; primer 239 and 267 wereemployed for quantification of CMV promoter/enhancer DNA methylated atposition 425. Samples were tested in triplicates. Templates #11, #62 and#01 were used as controls.

The qPCR was set up as reported in Example 5 by combining 5 μl templatewith 15 μl PCR master mix. The PCR conditions were as reported inExample 5. The primer annealing temperature was 58° C.

Methylation Specific qPCR with Bisulfite Treated Genomic DNA

Genomic DNA was extracted from CHO cell lines K18.1, G25-10, G25-17,G42-5 and 43-16 A10, cleaved with the enzyme DraI and deaminated bybisulfite treatment. Two microliters deaminated DNA diluted in 3 μlwater was used as template in real-time qPCR applying primer pair239/237 for amplification of total CMV promoter/enhancer strand A andprimer pair 239/267 for amplification of CMV promoter/enhancer strand Amethylated at position 425. Samples were tested in triplicates.Templates #11 and #62 were used as controls.

The PCR was set up and qPCR was performed as reported in Example 5. Theprimer annealing temperature was 58° C.

For a) and b) the fraction of promoter DNA methylated at position 425was calculated as follows:

mC=1.7^(Cp(t)-Cp(m)*)100  (Formula 7)

with

-   -   mC [%]: fraction of DNA methylated at position 425,    -   Cp(t): Cp value obtained with universal primer pair 239/237,    -   Cp(m): Cp value obtained with methylation-specific primer pair        239/267.

Both assay set-ups provided comparable results. The standard deviationwithin triplicates was higher without pre-amplification of the CMVpromoter strand A (FIGS. 8A and 8B). Methylation of position 425 of theCMV promoter nucleic acid in cell lines K18.1, G25-10, G25-17 and 43-16A10 was higher than the background of incomplete deamination, which hadbeen found to be approximately 1%.

Methylation in cell line G42-5 was below or maximum at background level.For cell line K18.1 the highest methylation was determined (more than60%).

Assay setting a) was performed with other universal and methylationspecific primer pairs. For the calculation of the fraction of DNAmethylated at position 425, the amplification efficiency for all primerpairs was assumed to be 2:

mC=2^(Cp(t)-Cp(m)*)100  (Formula 5′)

with

-   -   mC [%]: fraction of DNA methylated at position 425,    -   Cp(t): Cp value obtained with universal primer pair,    -   Cp(m): Cp value obtained with methylation specific primer pair.

Table 35 shows a summary of primer pair combinations that have beentested on either cloned DNA templates or on genomic DNA with or withoutpre-amplification of CMV promoter DNA.

TABLE 35 Primer pair combinations. position Combination universal 425specific 1 239 + 237 239 + 266 2 263 + 237 263 + 266 3 264 + 237 264 +266 4 239 + 237 239 + 267 5 263 + 237 263 + 267 6 264 + 237 266 + 267

EXAMPLE 8

Methylation of Human CMV Major-Immediate-Early Promoter/Enhancer andPrediction of Production Instability of Recombinant CHO Cell Line

CHO-K1 cells were transfected with a plasmid coding for a human IgG4antibody and stable clones were selected using the DHFR/MTX system. Highproducing parental clones were subcloned by limiting dilution. MTX waskept in the growth medium during the complete cell line generationprocess. 16 subclones from 10 parental clones were selected.

Selected cells were re-cultivated with 250 nM MTX. As soon as theyshowed stable growth, they were tested for long term productionstability over 60 to 80 generations in the presence and in the absenceof MTX. The relative alteration of the SPR over 60 generations wascalculated. Methylation of C435 was determined at the beginning of thestudy with cells grown with MTX and at the end of the study from cellsthat had been cultivated without MTX.

C425 methylation at start of the study was plotted against the relativealteration of the SPR in the presence (FIG. 11A) and in the absence ofMTX (FIG. 11B). The majority of clones with less than 5% methylation atC425 can be found in the fraction of stable clones (less than 40%decrease of SPR with or without MTX), whereas the majority of cloneswith more than 5% methylation at C425 clustered in the fraction ofinstable clones. This was independent of weather methylation wascorrelated with stability in the presence or in the absence of MTX (seealso Table 36. Most of the stable clones, that lose less than 20%productivity with MTX and less than 30% productivity without MTX,exhibit less than 5% C425 methylation.

TABLE 36 Correlation of methylation with stability in the presence or inthe absence of MTX (A) and plasmid copy number (B). number of cloneswith number of clones with methylation at C425 methylation at C425 A-16clones at start less than 5% at start more than 5% SPR_(rel)_End ≥ 60% 83 (cultivation in the presence of MTX) SPR_(rel)_End < 60% 1 4(cultivation in the presence of MTX) SPR_(rel)_End ≥ 60% 6 2(cultivation in the absence of MTX) SPR_(rel)_End < 60% 3 5 (cultivationin the absence of MTX) plasmid plasmid copy copy number number equalB-16 clones less than 10 to or more than 10 SPR_(rel)_End ≥ 60% 7 4(cultivation in the presence of MTX) SPR_(rel)_End < 60% 0 5(cultivation in the presence of MTX) SPR_(rel)_End ≥ 60% 6 2(cultivation in the absence of MTX) SPR_(rel)_End < 60% 1 7 (cultivationin the absence of MTX)

This finding shows that the determination of C425 methylation can beused as a predictive marker to determine the stability of polypeptideexpression in generated cell clones and thereby allowing the selectionof stable clones with stable productivity during cell line development.It has been found that C425 methylation of 5% or less is a suitablecriterion for the selection of stable cell clones.

It has further been found that two intermediately stable cell clones aswell as two very instable clones that were non-methylated at thebeginning of the study rise above 5% in methylation during stabilitytesting without MTX (see also FIG. 12 ). This shows that the fraction ofcell clones that are falsely predicted as stable (false negative cellclones) can be reduced by cultivating them for some time in the absenceof MTX before testing.

In order to confirm C425 methylation by a second method, we performedbisulfite sequencing with highly methylated clone 44-28 (FIG. 13 ). Thedegree of methylation at C425 was found to be 80%. This was consistentwith the result of methylation specific PCR, considering the variationof both assays. As with clones K18.1 and 43-16 A10 (FIG. 4 ), C425 wasmost often methylated among all CpG sites within the human CMVmajor-immediate-early promoter/enhancer DNA. Other methylation eventsclustered at the 5′ end and the 3′ end. The average degree ofmethylation at all sites was 18%.

EXAMPLE 9

Early Methylation Coincides with High Transgene Copy Numbers

The integrated copies of immunoglobulin light and heavy chain genes atthe beginning and at the end of stability testing were determined usinga multiplex qPCR assay based on the TaqMan principle. Two primer setsconsisting of a forward primer, a reverse primer and a hydrolysis probewere used: the one being specific for the human kappa chain gene, theother being specific for human gamma heavy chain genes. To allowdetermination of absolute copy numbers, the linearized expressionplasmid, which had been used for transfection, was used as a standard.Equal amplification efficiency of samples and standard were assured.

For qPCR, the LightCycler® 480 II system was employed (Roche DiagnosticsGmbH, Mannheim, Germany) and samples were prepared using theLightCycler® 480 Probes Master (Roche Diagnostics GmbH, Mannheim,Germany).

5 μl template solution containing 50 ng genomic DNA was combined with 15μl PCR master mix in the well of a 96-well microtiter plate. In case ofthe standard, the template solution contained 2.5×10⁷, 2.5×10⁶, 2.5×10⁵,2.5×10⁴ and 2.5×10³ copies of the corresponding linearized plasmid DNA.

15 μl PCR master mix comprised:

-   -   10 μl LightCycler® 480 Probes Master    -   1 μl forward primer #133 (10 pmol/μl)    -   1 μl reverse primer #132 (10 pmol/μl)    -   0.5 μl probe #166 (10 pmol/μl)    -   1 μl forward primer #178 (10 pmol/μl)    -   1 μl reverse primer #180 (10 pmol/μl)    -   0.5 μl probe #185 (10 pmol/μl)

The plate was sealed with a LightCycler® 480 sealing foil (RocheDiagnostics GmbH, Mannheim, Germany) and centrifuged at 1,500 g for 2minutes. Afterwards the plate was mounted into the LightCycler® 480system and subjected to qPCR. Each sample was tested in triplicate,standards were run in quadruplicate.

TABLE 37 PCR conditions were as follows: ramp No. of T t rate acqui-step cycles [° C.] [min:s] [° C. s-1] sition de-  1 95 10:00 4.40 —naturation real-Time denaturation 45 95 00:10 4.40 — PCR annealing 6000:05 2.20 — elongation 72 00:01 4.40 single cooling  1 37 01:00 2.2 

The collection and analysis of the data was performed using theLightCycler® 480 software version 1.5. Basically, mean Cp values of theplasmid standard dilutions were plotted against the respective gene copynumbers to generate a standard curve from which the number of transgenesin the sample was extrapolated.

The number transgenes per cell was calculated assuming that the averageDNA content per cell is jpg:

N _(e) =N _(s)/50000*6

-   -   N_(e): number of transgene copies per cell    -   N_(s): number of transgene copies in the sample

TABLE 38 Primer sequences. SEQ Primer Primer sequences ID no. (5′-> 3′)NO: 133 TCACAGAGCAGGACAGCAAG 26 132 GACTTCGCAGGCGTAGACTT 27 166(FAM)-AGCACCTACAGCCT 28 CAGCAGCACC-(BHQ1) 178 CGAACCGGTGACGGTGT 29 180GAGGGCACGGTCACCAC 30 185 (Cy5)-CACACCTTCCCGGC 31 TGTCCTACAG-(BHQ3)

FIG. 14 depicts the light chain gene copy numbers of methylated andnon-methylated cells before and after stability testing. Notsurprisingly, identical copy numbers were found the heavy chain genebecause the cells had been transfected with a double gene vectorcarrying both genes (data not shown). Early methylation i.e. methylationbefore stability testing was exclusively found with cells carrying morethan 10 transgene copies whereas some clones with less than 10 transgenecopies acquired methylation during stability testing. As a consequence,selecting clones with low transgene copy numbers before stabilitytesting equally enriches clones with stable productivity (Table 39).

TABLE 39 Correlation of transgene copies with stability in the presenceor in the absence of MTX. 16 clones Plasmid copies < 10 Plasmid copies ≥10 qP_(rel)_End + MTX ≥ 60% 7 4 qP_(rel)_End + MTX < 60% 0 5qP_(rel)_End − MTX ≥ 60% 6 2 qP_(rel)_End − MTX < 60% 1 7

We further observed that production instability was not generallyassociated with a loss of transgene copies. Clones 1A5-05, 1A5-21,1A5-24 and 2B1-02 lost transgene copies, 2B-13 was stable and 14-13 aswell as 14-23 increased in gene copies (see also FIG. 11B).

EXAMPLE 10

Identification of Present Histone Modifications Close to Human CMVMajor-Immediate-Early Promoter/Enhancer DNA by ChromatinImmunoprecipitation and Real-Time PCR

Chromatin Immunoprecipitation of CHO Cell Lines

The human CMV major-immediate-early promoter/enhancer fragment (SEQ IDNO: 01) used for the expression of antibody light and heavy chain isclose to DNA compacting histones (chromatin) in stable producer celllines. An accumulation of modified histones close to the human CMVmajor-immediate-early promoter/enhancer fragment will be measured withthe chromatin immunoprecipitation assay (ChIP).

Thirty-two CHO cell lines (samples H-1 to H-20 and T-1 to T-12, seeTable 311) were fixed in 3.7% formaldehyde for 10 min. at roomtemperature. Fixation was stopped in 10% glycine for 5 min. Cells werelysed for 10 min. on ice in Cell Lysis Buffer, centrifuged with 5,000rpm, 4 min. at 4° C. Pellets were resuspended in Nuclei Lysis Buffer andchromatin was sonicated to an average fragment size of 200-500 bp withBranson Sonifier B15. Concentrations of chromatin fragments weredetermined with Pierce® BCA Protein Assay Kit (Thermo Scientific,Rockford, USA). 2.5 μg-100 μg chromatin fragments were added to 1.5volumes IP Dilution Buffer containing 3 μl-12 μl antibody and incubatedovernight at 4° C. while rotating with 25 rpm.

Protein A Agarose beads (Roche Diagnostics GmbH, Mannheim, Germany) wereblocked in IP Dilution Buffer with 100 μg/ml salmon sperm DNA(Invitrogen, Carlsbad, U.S.A) and 5 mg/ml BSA (Bovine Serum Albumin,Roche Diagnostics GmbH, Mannheim, Germany) overnight at 4° C.Antibody-chromatin precipitates were purified by incubation in blockedAgarose beads for 1 hour, followed by incubation for 5 min at 4° C. in2× Low, 1× High salt, 1×LiCl and 1×TE wash buffer each. Antibodies wereeluted from the Agarose beads with 200 μl IP Elution buffer, for 30 min.at 65° C. 0.5 μl RNAse DNAse free (Roche Diagnostics GmbH, Mannheim,Germany) were added to digest RNA at 37° C. for 30 minutes. For proteindigestion NaCl (final concentration 0.2 M) and Proteinase K (finalconcentration 100-200 μg/ml) (Roche Diagnostics GmbH, Mannheim, Germany)were added and solution was incubated for 1.5 hours at 65° C. DNA wasrecovered with Roche PCR Purification Kit in 150 μl PCR grade H₂O (RocheDiagnostics GmbH, Mannheim, Germany).

TABLE 40 Buffer for Chromatin Immunoprecipitation (ChIP). CHIP Celllysis buffer LiCl wash buffer 20 mM Tris-HCl pH 8.0 0.25M LiCl 85 mM KCl(Villagra et al. CHIP) 0.5% NP40 1% NP40 (octylphenolpolyethoxy 1% deoxycholate ethanol) (deoxycholic acid) double distilled water 1 mM EDTANuclei Lysis buffer 20 mM Tris, pH 8.0 50 mM Tris-HCl pH 8.0 doubledistilled water 10 mM EDTA pH 8.0 TE buffer 1% SDS 10 mM Tris-HCl pH 8.0double distilled water 1 mM EDTA 1 tablet Roche (IP) Elution bufferComplete per 10 ml 50 mM NaHCO3 Low salt wash buffer 1% SDS 0.1% SDSdouble distilled water 1% Triton X 100 IP Dilution buffer 2 mM EDTA 1.1%Triton X 100 20 mM Tris-CL pH 8.0 1.2 mM EDTA 150 mM NaCl 16.7 mMTris-HCl pH 8.1 (8.0) double distilled water High salt wash buffer 0.1%SDS 1% Triton X 100 2 mM EDTA 20 mM Tris-CL pH 8.0 500 mM NaCl 167 mMNaCl H2O

TABLE 41 Antibodies specific for histone or histone modifications. ChIPTarget name company grade host cat. no. lot H3K4me3 ChIPAb+ Millipore vrabbit 17-614 Trimethyl-histone monoclonal ng1848343 H3 (Lys4) H3K27me3ChIPAb+ Millipore v rabbit 17-622 Trimethyl-histone polyclonaldam1850974 H3 (Lys27) H3K9me3 Anti-histone H3 Abcam v rabbit ab8898(trimethyl K9) polyclonal gr58934-l antibody H3 Anti-histone H3 Abcam vrabbit ab1791 antibody polyclonal GR103799 H3ac Anti-acetyl-histoneMillipore v rabbit 06-599 H3 polyclonal 2068182 ChIP target name companygrade host cat. no .lot H3K4me3 histone H3K4me3 activemotif √ rabbit39915 antibody (pAb) Polyclonal 14013006 H3ac Anti-acetyl-histoneMillipore √ rabbit 06-599 H3 Antibody polyclonal 2153150 H3K9me3 ChIPAb+Millipore √ polyclonal 17-625 Trimethyl-histone 2190636 H3 (Lys9)H3K27me3 ChIPAb+ Millipore v rabbit 17-622 Trimethyl-histone Polyclonal2034145 H3 (Lys27) H3 Anti-histone H3 Abcam √ Rabbit ab1791 antibodypolyclonal GR135321

Quantitative Real Time PCR of ChIP DNA

Real time quantitative PCR was used to detect an accumulation ofspecific histone modifications on the human CMV major-immediate-earlypromoter/enhancer fragment (SEQ ID NO: 01) in CHO cell lines bymeasuring the relative amount of the human CMV major-immediate-earlypromoter/enhancer fragment per chromatin immunoprecipitation.

Genomic DNA from untreated Input Sample (IS) and antibody purifiedchromatin fragments were amplified with real time quantitative PCR usingLightCycler 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim,Germany) using the LightCycler 480 II system (Roche Diagnostics GmbH,Mannheim, Germany). To quantify the amount of human CMVmajor-immediate-early promoter/enhancer fragment (SEQ ID NO: 01)specific primer for possible reference genes and for the human CMVmajor-immediate-early promoter/enhancer fragment were used.

TABLE 42 Primer sequences for real time quantitative PCR. SEQ ID PrimerNO: NO: Target Sequence 32 396 hCMV-MIE forward TACATCAATGGGCGTG 1 GATA33 397 hCMV-MIE reverse AAGTCCCGTTGATTTT 1 GGTG 34 386 Gusb forward 1CAGGGTGGGATGCTCTTC 35 387 Gusb reverse 1 GCCGGTTTTCCGAGAAGT 36 431Eifi forward 1 GTTCCCGGCACTGACACT 37 432 Eif3i reverse 2ACTTGATCTGCGTGATG GAC 38 484 Fox2a forward 1 ATCACCCGTACTGCTGC TCT 39485 Fox2a reverse GAGGCTTCTGGGGATCT CTT 40 468 Gata5 forward 1CACCTACCCCATCCTGT CTG 41 469 Gata5 reverse 1 GAGGAGGTGAAGGCAAA GTCT 42388 Rho forward 1 AGCCTCGGTCTCTATTG ACG 43 389 Rho reverse 1CGTTGGAGAAGGGCACA TAA

2.5 μl eluted DNA fragments, 1 μl (10 pmol/μl) of forward primer 396(SEQ ID NO: 32) and 1 μl (10 pmol/μl) of reverse primer 397 (SEQ ID NO:33) for the human CMV major-immediate-early promoter/enhancer fragment(SEQ ID NO: 01), 5 μl LightCycler 480 SYBR Green I Master and nucleasefree water were mixed up to a total volume of 10 μl. Reference geneswere quantified on the same plate. The mix was applied to 384 well plate(Roche Diagnostics GmbH, Mannheim, Germany).

The plate was sealed with a LightCycler® 480 sealing foil (RocheDiagnostics GmbH, Mannheim, Germany) and centrifuged at 1,200 g for 3minutes. Afterwards the plate was mounted into the LightCycler® 480system and subjected to real time qPCR. Each sample was tested intriplicate.

TABLE 43 PCR conditions were as follows: ramp No. of T t rate acqui-step cycles [° C.] [min:s] [° C. s-1] sition de-  1 95 15:00 4.40 —naturation real-time denaturation 45 95 00:15 4.40 — PCR annealing 6000:10 2.20 single elongation 72 00:15 4.40 — melting denaturation  1 9500:05 4.40 — curve annealing 60 01:00 2.20 — melting 90 00:00 0.11contin- uous cooling  1 40 00:30 2.2 

The collection and analysis of the data was done with the LightCycler®480 software version 1.5.

EXAMPLE 11

Relative Quantification of Human CMV Major-Immediate-EarlyPromoter/Enhancer Fragment on Histone Modifications in Real-Time PCR

Stability of Reference Genes

In this example the stability of potential reference genes for specifichistone modifications was calculated with the program “Normfinder”. Themodel and statistical framework underlying “NormFinder” are described inAndersen, C. L., et al. Cancer Res. 64 (2004) 5245-5250.

The “NormFinder add on” for excel provides the stability value for eachgene, which is a direct measure for the estimated expression variation.The gene with the smallest stability value is the most stable gene. Theinput data is supposed to be on a linear scale, thus the Percent InputMethod was used to linearize scale expression quantities using thefollowing formula:

% ofInputSample=100*2^(ΔCq(InputSample-ChIPSample))   (Formula 9)

Five genes (see Table 44) were tested with “Normfinder”. Twenty samples(H-1 to H20) in three biological replicates were investigated for thestability of the histone 3 acetylation, histone 3 lysine 4 three-foldmethylation and histone 3 close to the human CMV major-immediate-earlypromoter/enhancer fragment and the reference genes.

TABLE 44 Stability values of possible reference genes and the human CMVmajor-immediate-early promoter/enhancer fragment. H3ac % of input sampleH3 % of input sample H3K4me3 % of Input Sample Gene name Stability valueGene name Stability value Gene name Stability value hCMV-MIE 10.729hCMV-MIE 0.571 hCMV-MIE 5.399 Eif3i 0.160 Eif3i 0.138 Eif3i 1.792 Fox2a0.491 Fox2a 0.181 Fox2a 1.711 Gata5 0.481 Gata5 0.141 Gusb 1.680 Gusb0.093 Gusb 0.136

Reference gene Gusb is more stable in chosen ChIP conditions than theother controls. Gusb was used as reference gene for the normalization ofhistone 3 acetylation, histone 3 lysine 4 three-fold methylation andhistone 3 levels.

Estimation of Amplification Efficiency of Primer Pairs with LinRegPCRSoftware

In this example the amplification efficiency of the primer pair 396/397for the human CMV major-immediate-early promoter/enhancer fragment andof the primer pair 386/387 for reference gene Gusb were estimated withthe LinRegPCR software (www.hartfaalcentrum.nl) for samples H-1 to H20.The program determines baseline fluorescence and does a baselinesubtraction. Then a Window-of-Linearity is set and PCR efficiencies persample are calculated (see Ramakers, et al., NeuroSci Lett. 2003;Ruijter et al., Nucleic Acids Research 2009).

Eff=10^(slope)  (Formula 11)

The individual PCR efficiency (Eff) is deduced from the slope of thelinear regression line and is defined a fold increase per cycle. It canrange between 1 and 2. An efficiency of 2 represents a perfect doublingof the amplicon in each cycle.

TABLE 45 Amplification efficiency of reference Gusb primer pair andtarget hCMV-MIE primer pair. Amplification efficiency: Primer pair Meanefficiency Stdev. hCMV MIE 396/7 1.911 0.0068 Gusb 386/7 1.930 0.0068

Primer pairs 396/397 and 386/387 yielded stable and comparableamplification efficiencies with 20 samples and 3 biological replicatesirrespective of whether ChIP input samples were measured or DNAprecipitated with antibodies against histone 3, acetylated histone 3 orhistone 3 lysine 4 three-fold methylation. Primer pairs 396/397 and386/387 were used for the relative quantification of histone 3acetylation, histone 3 lysine 4 three-fold methylation and histone 3levels close to human CMV major-immediate-early promoter/enhancerfragment relative to histone 3 and histone 3 modification levels closeto reference gene Gusb.

Delta Delta Cq Method for Relative Quantification of HistoneModification Relative to the Level of Histone

The relative quantification of histone 3 acetylation, histone 3 lysine 4three-fold methylation and histone 3 levels close to human CMVmajor-immediate-early promoter/enhancer fragment were estimated with theLivak method, also known as delta delta Cq (ΔΔCq) method.

The method can be used for relative quantification of a target regardingto a reference. In this example the relative quantification of histone 3acetylation, histone 3 lysine 4 three-fold methylation and histone 3levels close to human CMV major-immediate-early promoter/enhancerfragment were normalized to the histone 3 modification and the histone 3levels close to reference gene Gusb in two steps.

First delta Cq was calculated as follows:

ΔCq=inputsample−sample  (formula 12)

Delta Cq (ΔCq) is the difference between quantification cycles (Cq) ofuntreated (input sample) and treated (ChIP sample) condition of the samesample, amplified with the same primer pair. The delta Cq is used todepict a value relative to the untreated condition within the samesample. To compare different samples, the delta delta Cq method wasused.

$\begin{matrix}{{{{\Delta\Delta}{Cq}}--} > \frac{{ratio} = {2^{\Delta{Cq}}{target}^{({{control} - {sample}})}}}{2^{\Delta{Cq}}{ref}^{({{control} - {sample}})}}} & \left( {{formula}13} \right)\end{matrix}$

In this example delta delta Cq is the difference between delta Cq of thetarget hCMV MIE and delta Cq of the reference Gusb for all samples H-1to H-20. Relative amounts of DNA determined using delta delta Cq valuescan be compared between samples.

Finally levels of histone 3 acetylation and histone 3 lysine 4three-fold methylation were normalized to the level of histone 3 for the20 CHO cell lines H-1 to H-20 in three biological replicates. Theresults with the standard deviation are displayed in FIGS. 15 and 16 .

EXAMPLE 12

Histone 3 Acetylation and Histone 3 Lysine 4 Three-Fold MethylationClose to Human CMV Major-Immediate-Early Promoter/Enhancer Fragment asWell as Methylation of C425 of Human CMV Major-Immediate-EarlyPromoter/Enhancer Fragment Correlate with Long-Term Stability

The relative acetylation and the lysine 4 threefold methylation levelsrelative to the level of histone 3 (H3ac/H3 & H3K4me3/H3) close to humanCMV major-immediate-early promoter/enhancer fragment as well as thepercentage of methylation of C425 of human CMV major-immediate-earlypromoter/enhancer fragment (mC-425[%]) were investigated in a fit modelwith the personality “Standard least squares” and the emphasis “effectleverage model” to detect effects influencing the long term stability ofthe producer cell lines H-1 to H-20. For calculation the JMP softwareversion 10 (SAS, Boeblingen, Germany) was used.

The histone modification values and the percentage of C-425 methylationof samples H-1 to H-20 were fed as an effect into the modelindividually. The percentual alteration of specific production rate(delta SPR) after 60 generations was fed as response. Resulting leverageplots are displayed in FIGS. 17 to 19 . The number of parametersassociated with the effect (Nparm) and the degrees of freedom are 1.

RSquare (RSq) estimates the proportion of variation in the response thatcan be attributed to the model rather than to random error. An R² closerto 1 indicates a better fit. An R² closer to 0 indicates that the fitpredicts the response no better than the overall response mean. RootMean Square Error (RMSE) estimates the standard deviation of the randomerror. It is the square root of the Mean Square of Error in the Analysisof Variance report. Prob>F lists the p-value for the Effect test, whichinforms about the significance of an effect.

TABLE 46 P-values of fed effects H3K4me3/H3 and H3ac/H3 calculated ineffect test. Calculated in three biological replicates for samples H.Prob > F lists the p-value for the Effect test Selection conditionEffect With MSX Without MSX H3K4me3/H3 0.0102 * 0.889 H3ac/H3 <0.0001 *0.1449 mC-425 [%] 0.0898 0.0041 * * values of 0.05 or less wereconsidered evidence of significant effect in the model.

Table 46 shows the significance of mC-425[%] under (−) MTX conditions.The effect of H3ac/H3 and H3K4-me3/H3 are significant under (+) MSXcondition.

Actual delta SPR values were plotted against predicted values inleverage plots, regarding to the effect and the selection condition over60 generations (FIG. 17-19 ). This Actual by Predicted plot shows howwell the model fits the data. The lowest p-values were detected for theH3ac/H3 effect with selection agent (FIG. 18 ) followed by mC 425[%]effect without selection agent (FIG. 19 ).

Outlier Analysis Comparing Conditions with and without Selection AgentMSX for Delta SPR Values of Samples H-1 to H-20

The leverage plots of the condition without selection agent ((−) MSX)have remarkably aberrant values for delta SPR in sample H-18. Thereforan outlier analysis showing Jackknife Distances was done (FIG. 20 ).

This verifies that the increase of delta SPR in sample H-18 under thecondition (−) MSX is abnormal compared to all other samples. Thereforeleverage analyses were repeated without sample H-18. The calculatedp-values of the effect test for 19 samples H-1 to H-17 and H-19 to H-20are displayed in Table 47.

TABLE 47 P-values of fed effects H3K4me3/H3 and H3ac/H3 calculated ineffect test. P-values calculated of 19 samples (H-1 to H-17, H-19, H-20)in three biological replicates. Prob > F lists the p-value for theEffect test Selection condition Effect With MSX Without MSX H3K4me3/H30.0136 * 0.3464 H3ac/H3 <0.0001 * 0.0001 * mC-435 [%] 0.1171 0.0908 *Values of 0.05 or less were considered evidence of significant effect inthe model.

Table 47 shows high significance of H3ac/H3 in both selectionconditions. The effect of H3K4-me3/H3 is significant in condition (+)MSX.

Actual delta SPR values were plotted against predicted values inleverage plots, regarding the effect and the selection condition over 60generations (FIG. 20-22 ). The Actual by Predicted plots show how wellthe model fits the data. The best fit was observed for the H3ac/H3effect (FIG. 21 ).

EXAMPLE 13

Prediction of Production Instability in Recombinant CHO Cell Lines byUsing Histone Modifications Close to Human CMV Major-Immediate-EarlyPromoter/Enhancer and Methylation of C425 of Human CMVMajor-Immediate-Early Promoter/Enhancer Fragment as Marker.

Increased Mean Delta SPR of Project H Samples by Rejecting Bad ProducerCell Lines with Filter Settings

As reported in Example 12, values of outlier sample H-18 were excludedfrom further investigations of establishing prediction marker.

Levels of histone 3 acetylation relative to the level of histone 3 closeto human CMV major-immediate-early promoter/enhancer fragment andpercentage of C-425 methylation of human CMV major-immediate-earlypromoter/enhancer fragment were investigated as prediction markers forproduction stability with and without selection agent.

As described in Osterlehner et al. filter for mC-425[%] values was setto 5%. Values above 5% were rejected to increase the mean delta SPR ofremaining samples.

For H3ac/H3 values of samples H-1 to H-17, H-19 and H-20 a decision treewas calculated with jmp software. Best split node and therefor bestfilter was calculated with the LogWorth statistic. The LogWorth iscalculated as:

−log 10(p-value)  (Formula 14)

where the adjusted p-value is calculated in a complex manner that takesinto account the number of different ways splits can occur. Thiscalculation is very fair compared to the unadjusted p-value, whichfavors X's with many levels, and the Bonferroni p-value, which favorsX's with small numbers of levels (white paper: “Monte Carlo Calibrationof Distributions of Partition Statistics”).

The best split under (+) MSX condition for samples H-1 to H-17, H-19 andH-20 was 0.58 histone 3 acetylation relative to the level of histone 3(H3ac/H3). To reduce number of false negative samples the filter was setto the lower value of A>0.5 H3ac/H3.

Both filters were used separate and in combination. The means of deltaSPR at conditions with or without selection agent MSX were calculatedfor each positive gate and compared to the unfiltered mean (Table 48).

TABLE 48 Calculated means of delta SPR with and without selection agentand under filter conditions A > 0.5 H3ac/H3 and B < 5% mC-425 [%] PSP.Values were calculated for samples H-1 to H-17, H-19 and H-20 referringto filter settings. Mean ΔSPR Mean ΔSPR n (+) MSX (−) MSX unfiltered 56−0.39 −0.55 A > 0.5 H3ac/H3 23 (41%) −0.15 −0.38 B < 5% mC-425[%] PSB 27(48%) −0.24 −0.45 Combined filter (B ∩ A) 20 (36%) −0.13 −0.36

Each filter can increase the mean delta SPR compared to the unfilteredcondition. The intersection of filter A and B increases the mean deltaSPR of filter A>0.5 H3ac/H3. For visualization of altered delta SPRvalues, histograms of each condition and filter are displayed in FIG. 23.

Established Filter Settings of Project H were Adopted to Project T toConfirm Positive Effects

Twelve cell lines (T-1 to T-12) were also analyzed with filter settingsas determined above (see Table 49). For visualization of altered deltaSPR values, histograms of each condition and filter are displayed inFIG. 25 .

TABLE 49 Calculated means of delta SPR with and without selection agent.Values were calculated for 12 samples T-1 to T-12 referring to filtersettings. Mean ΔSPR Mean ΔSPR n (+) MTX (−) MTX unfiltered 36 −0.29−0.41 A > 0.5 H3ac/H3 7 (19%) −0.19 −0.32 B < 5% mC-425[%] PSB 24 (67%) −0.28 −0.43 Combined filter (B ∩ A) 7 (19%) −0.19 −0.32

Observed increase of mean delta SPR compared filtered to unfilteredsamples under (−) MTX condition.

Using the filter setting of A>0.5 H3ac/H3 results in an increase of meandelta SPR compared to unfiltered data.

This shows that the histone 3 acetylation relative to the level ofhistone 3 close to human CMV major-immediate-early promoter/enhancerfragment is a valuable prediction marker.

1. A method for selecting a cell clone/cell line from cells thatcomprise a nucleic acid comprising a structural gene encoding apolypeptide operably linked to a promoter nucleic acid that has thenucleic acid sequence of SEQ ID NO: 01 comprising the following steps:a) determining a ratio of the level of histone 3 acetylation relative tothe level of histone 3 close to the promoter nucleic acid, wherein closeto the promoter nucleic acid is within several hundred base pairsupstream of the promoter, for a first multitude of the cells, b)determining a methylation frequency of the CpG-site at position 425 ofSEQ ID NO: 01 for a second multitude of the cells, and c) selecting acell clone/cell line that has the ratio as determined in step a) of morethan 0.5 and that has the methylation frequency as determined in step b)of less than 5%.
 2. The method according to claim 1 wherein, in step(a), the ratio is an average of the level of histone 3 acetylationrelative to the level of histone 3 close to the promoter nucleic acid,determined for at least 10 cells obtained from culturing a candidatecell clone/cell line, and in step (ii), the methylation frequency is anaverage methylation frequency of the CpG-site at position 425 of SEQ IDNO: 01, determined for at least 10 cells obtained from culturing thecandidate cell clone/cell line.
 3. The method according to claim 1wherein the polypeptide is an antibody light chain and wherein step a)further comprises: determining the copy number of stably integratedlight chain expression cassettes, and wherein step c) further comprises:selecting the cell clone/cell line to also have a copy number of stablyintegrated light chain expression cassettes of 10 or less.
 4. The methodaccording to claim 1, wherein the first multitude of cells and thesecond multitude of cells are the same multitude of cells.
 5. The methodaccording to claim 1, wherein the determining of the level of histone 3acetylation relative to the level of histone 3 comprises the followingsteps: 1) isolating chromatin from a candidate cell clone/cell line, 2)treating a first aliquot of the chromatin with a histone 3 acetylationspecific antibody or a histone 3 specific antibody to form a firstantibody-chromatin precipitate, and treating a second aliquot of thechromatin with a histone 3 specific antibody to form a secondantibody-chromatin precipitate, 3) amplifying genomic DNA from a thirdnon-treated aliquot of the chromatin and from the first treated aliquotand from the second treated aliquot with real time quantitative PCR, and4) determining with the result obtained in step 2) and in step 3) thelevel of histone 3 acetylation relative to the level of histone
 3. 6.The method according to claim 1, wherein the determining of themethylation frequency comprises the following steps: 1) isolating theDNA from a candidate cell clone/cell line, 2) performing for theisolated DNA a methylation specific polymerase chain reaction, and 3)determining with the results obtained in step 2) the methylationfrequency.
 7. The method according to claim 6, wherein in step 2) amethylation specific primer and a universal primer are used.
 8. Themethod according to claim 7, wherein the universal primers have thesequence of SEQ ID NO: 09 and 11 and the methylation specific primershave the sequence of SEQ ID NO: 11 and
 18. 9. The method according toclaim 1, wherein the level of histone 3 acetylation relative to thelevel of histone 3 is normalized to a reference gene.
 10. The methodaccording to claim 9, wherein the reference gene is Gusb.
 11. The methodaccording to claim 1, wherein the second multitude of cells comprises atleast one cell also in the first multitude of cells.
 12. The methodaccording to claim 11, wherein the second multitude of cells comprisesmore than one cell also in the first multitude of cells.
 13. The methodaccording to claim 1, wherein the cell clone/cell line is a CHO cellclone/cell line.
 14. The method according to claim 1, wherein thepolypeptide is an antibody or an antibody fragment or an antibodyconjugate.
 15. The method according to claim 14, wherein the antibody isa bispecific antibody. 16-17. (canceled)
 18. The method according toclaim 1, wherein close to the promoter nucleic acid is within 200 basepairs upstream of the promoter.
 19. The method according to claim 18,wherein close to the promoter nucleic acid is within 200 base pairsupstream of a transcription start site of the promoter.
 20. The methodaccording to claim 1, wherein the method comprises prior to step a) thefollowing steps: x) providing a mammalian, non-human cell, and y)transfecting the provided cell with a nucleic acid, which comprises astructural gene encoding a polypeptide operably linked to a promoternucleic acid that has the nucleic acid sequence of SEQ ID NO:01.